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1.
Cell ; 184(4): 983-999.e24, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33606986

RESUMO

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.


Assuntos
Interleucina-12/química , Interleucina-12/metabolismo , Células Matadoras Naturais/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Imunidade , Interleucina-12/agonistas , Subunidade p40 da Interleucina-12/química , Subunidade p40 da Interleucina-12/metabolismo , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/patologia , Estrutura Quaternária de Proteína , Receptores de Interleucina/ultraestrutura , Receptores de Interleucina-12/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
2.
J Biol Chem ; 295(33): 11486-11494, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32532817

RESUMO

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.


Assuntos
Antígenos de Neoplasias/imunologia , Antígeno HLA-A2/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Antígenos de Neoplasias/química , Cristalografia por Raios X , Antígeno HLA-A2/química , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química
3.
Nature ; 527(7578): 323-8, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26536114

RESUMO

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.


Assuntos
Antibacterianos/farmacologia , Bacteriemia , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Espaço Intracelular/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologia , Animais , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Portador Sadio/tratamento farmacológico , Portador Sadio/microbiologia , Desenho de Fármacos , Feminino , Imunoconjugados/química , Espaço Intracelular/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Testes de Sensibilidade Microbiana , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fagossomos/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade , Vancomicina/uso terapêutico
4.
Nature ; 515(7528): 572-6, 2014 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-25428506

RESUMO

Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, ∼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.


Assuntos
Exoma/genética , Fenômenos Imunogenéticos/genética , Espectrometria de Massas , Mutação , Neoplasias/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Imunidade Celular/imunologia , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neoplasias/imunologia , Peptídeos/genética , Estrutura Terciária de Proteína
5.
Bioorg Med Chem Lett ; 29(12): 1497-1501, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31000154

RESUMO

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.


Assuntos
Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Humanos , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Relação Estrutura-Atividade
6.
Bioorg Med Chem Lett ; 29(12): 1522-1531, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30981576

RESUMO

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).


Assuntos
Dermatite Atópica/genética , Interleucina-13/metabolismo , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Humanos , Transdução de Sinais
7.
PLoS Pathog ; 12(6): e1005702, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27351973

RESUMO

Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness.


Assuntos
Anticorpos Neutralizantes/imunologia , Resistência Microbiana a Medicamentos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Evasão da Resposta Imune/imunologia , Vírus da Influenza A/imunologia , Animais , Anticorpos Antivirais/imunologia , Western Blotting , Cães , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Influenza Humana/imunologia , Células Madin Darby de Rim Canino , Camundongos , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Reação em Cadeia da Polimerase
8.
Proc Natl Acad Sci U S A ; 111(22): 8025-30, 2014 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-24843152

RESUMO

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.


Assuntos
Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Janus Quinase 3/metabolismo , Neoplasias/enzimologia , Transdução de Sinais/fisiologia , TYK2 Quinase/metabolismo , Animais , Linhagem Celular , Cristalografia por Raios X , Dimerização , Ativação Enzimática/fisiologia , Humanos , Insetos/citologia , Janus Quinase 1/genética , Janus Quinase 2/genética , Janus Quinase 3/genética , Mutação , Neoplasias/genética , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , TYK2 Quinase/química , TYK2 Quinase/genética
9.
Mol Cell ; 31(5): 737-48, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18775332

RESUMO

gp130 is a shared receptor for at least nine cytokines and can signal either as a homodimer or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here, we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-Ralpha). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6Ralpha hexameric complex, CNTF/CNTF-Ralpha heterodimerizes gp130 and LIF-R via noncooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic analysis of the full-length gp130/LIF-R/CNTF-Ralpha/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the "tall" class of gp130 family cytokine receptor complexes including LIF, IL-27, IL-12, and others.


Assuntos
Receptor gp130 de Citocina/química , Complexos Multiproteicos/química , Estrutura Quaternária de Proteína , Receptores de OSM-LIF/química , Transdução de Sinais/fisiologia , Animais , Fator Neurotrófico Ciliar/química , Fator Neurotrófico Ciliar/genética , Fator Neurotrófico Ciliar/metabolismo , Cristalografia por Raios X , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/ultraestrutura , Receptor do Fator Neutrófico Ciliar/química , Receptor do Fator Neutrófico Ciliar/genética , Receptor do Fator Neutrófico Ciliar/metabolismo , Receptores de OSM-LIF/genética , Receptores de OSM-LIF/metabolismo , Termodinâmica
10.
J Biol Chem ; 288(37): 26926-43, 2013 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-23897821

RESUMO

Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/química , Histonas/química , Acetilação , Benzamidas/química , Ligação Competitiva , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/química , Concentração Inibidora 50 , Cinética , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Piridinas/química , Transcrição Gênica , Vorinostat
11.
Nat Chem Biol ; 8(12): 990-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23103943

RESUMO

Cytokines dimerize their receptors, with the binding of the 'second chain' triggering signaling. In the interleukin (IL)-4 and IL-13 system, different cell types express varying numbers of alternative second receptor chains (γc or IL-13Rα1), forming functionally distinct type I or type II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A type I receptor-selective IL-4 'superkine' with 3,700-fold higher affinity for γc was three- to ten-fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 more potently activated cells expressing the type II receptor and induced differentiation of dendritic cells from monocytes, implicating the type II receptor in this process. Superkines showed signaling advantages on cells with lower second chain numbers. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain numbers can be exploited to redirect cytokines toward distinct cell subsets and elicit new actions, potentially improving the selectivity of cytokine therapy.


Assuntos
Citocinas/fisiologia , Interleucina-4/análogos & derivados , Interleucina-4/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos/farmacologia , Interleucina-4/química , Mutação/fisiologia , Fenótipo , Fosforilação , Engenharia de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-4/efeitos dos fármacos , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
12.
Med ; 5(2): 132-147.e7, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38272035

RESUMO

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.


Assuntos
Escleroderma Sistêmico , Fator de Crescimento Transformador beta3 , Humanos , Fator de Crescimento Transformador beta3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Fibrose , Escleroderma Sistêmico/tratamento farmacológico , Isoformas de Proteínas/metabolismo
13.
Bioorg Med Chem Lett ; 23(21): 5923-30, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24042009

RESUMO

A highly ligand efficient, novel 8-oxo-pyridopyrimidine containing inhibitor of Jak1 and Jak2 isoforms with a pyridone moiety as the hinge-binding motif was discovered. Structure-based design strategies were applied to significantly improve enzyme potency and the polarity of the molecule was adjusted to gain cellular activity. The crystal structures of two representative inhibitors bound to Jak1 were obtained to enable SAR exploration.


Assuntos
Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Humanos , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade
14.
Bioorg Med Chem Lett ; 23(12): 3592-8, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23642482

RESUMO

The identification of a novel fused triazolo-pyrrolopyridine scaffold, optimized derivatives of which display nanomolar inhibition of Janus kinase 1, is described. Prototypical example 3 demonstrated lower cell potency shift, better permeability in cells and higher oral exposure in rat than the corresponding, previously reported, imidazo-pyrrolopyridine analogue 2. Examples 6, 7 and 18 were subsequently identified from an optimization campaign and demonstrated modest selectivity over JAK2, moderate to good oral bioavailability in rat with overall pharmacokinetic profiles comparable to that reported for an approved pan-JAK inhibitor (tofacitinib).


Assuntos
Janus Quinase 1/antagonistas & inibidores , Piridinas/farmacologia , Animais , Cristalografia por Raios X , Janus Quinase 1/química , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/química , Cinética , Modelos Moleculares , Piridinas/química , Pirróis/química , Pirróis/farmacologia , Ratos
15.
Bioorg Med Chem Lett ; 22(24): 7627-33, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23107482

RESUMO

Herein we describe our successful efforts in obtaining C-2 substituted imidazo-pyrrolopyridines with improved JAK1 selectivity relative to JAK2 by targeting an amino acid residue that differs between the two isoforms (JAK1: E966; JAK2: D939). Efforts to improve cellular potency by reducing the polarity of the inhibitors are also detailed. The X-ray crystal structure of a representative inhibitor in complex with the JAK1 enzyme is also disclosed.


Assuntos
Descoberta de Drogas , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Imidazóis/química , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Masculino , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Piridinas/administração & dosagem , Piridinas/química , Pirróis/administração & dosagem , Pirróis/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
16.
Science ; 376(6589): 163-169, 2022 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-35271300

RESUMO

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 valine→phenylalanine (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.


Assuntos
Janus Quinase 1 , Receptores de Citocinas , Domínios de Homologia de src , Regulação Alostérica , Microscopia Crioeletrônica , Ativação Enzimática , Humanos , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Mutação , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Fosforilação , Multimerização Proteica , Receptores de Citocinas/química , Fatores de Transcrição STAT/metabolismo
17.
Mol Cancer Ther ; 21(6): 974-985, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35364611

RESUMO

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.


Assuntos
Anticorpos Biespecíficos , Neoplasias Colorretais , Imunoglobulinas , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoglobulinas/imunologia , Camundongos , Instabilidade de Microssatélites , Linfócitos T/imunologia
18.
Nat Chem Biol ; 5(7): 469-78, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19465933

RESUMO

MARTX toxins modulate the virulence of a number of Gram-negative Vibrio species. This family of toxins is defined by the presence of a cysteine protease domain (CPD), which proteolytically activates the Vibrio cholerae MARTX toxin. Although recent structural studies of the CPD have uncovered a new allosteric activation mechanism, the mechanism of CPD substrate recognition or toxin processing is unknown. Here we show that interdomain cleavage of MARTXVc enhances effector domain function. We also identify the first small-molecule inhibitors of this protease domain and present the 2.35-A structure of the CPD bound to one of these inhibitors. This structure, coupled with biochemical and mutational studies of the toxin, reveals the molecular basis of CPD substrate specificity and underscores the evolutionary relationship between the CPD and the clan CD caspase proteases. These studies are likely to prove valuable for devising new antitoxin strategies for a number of bacterial pathogens.


Assuntos
Toxina da Cólera/química , Toxina da Cólera/metabolismo , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Vibrio cholerae/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Western Blotting , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Espectroscopia de Infravermelho com Transformada de Fourier , Especificidade por Substrato , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/enzimologia
20.
Nat Rev Drug Discov ; 20(1): 39-63, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33077936

RESUMO

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.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Autoimunidade/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/química , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia
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