RESUMO
Defective host defenses later in life are associated with changes in immune cell activities, suggesting that age-specific considerations are needed in immunotherapy approaches. In this study, we found that PD-1 and CTLA4-based cancer immunotherapies are unable to eradicate tumors in elderly mice. This defect in anti-tumor activity correlated with two known age-associated immune defects: diminished abundance of systemic naive CD8+ T cells and weak migratory activities of dendritic cells (DCs). We identified a vaccine adjuvant, referred to as a DC hyperactivator, which corrects DC migratory defects in the elderly. Vaccines containing tumor antigens and DC hyperactivators induced T helper type 1 (TH1) CD4+ T cells with cytolytic activity that drive anti-tumor immunity in elderly mice. When administered early in life, DC hyperactivators were the only adjuvant identified that elicited anti-tumor CD4+ T cells that persisted into old age. These results raise the possibility of correcting age-associated immune defects through DC manipulation.
Assuntos
Linfócitos T CD4-Positivos , Células Dendríticas , Camundongos Endogâmicos C57BL , Células Dendríticas/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Camundongos , Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunoterapia/métodos , Vacinas Anticâncer/imunologia , Feminino , Neoplasias/imunologia , Neoplasias/terapia , Receptor de Morte Celular Programada 1/metabolismo , Antígeno CTLA-4/metabolismo , Movimento Celular , Antígenos de Neoplasias/imunologiaRESUMO
Homologous recombination deficiency (HRD) is prevalent in cancer, sensitizing tumor cells to poly (ADP-ribose) polymerase (PARP) inhibition. However, the impact of HRD and related therapies on the tumor microenvironment (TME) remains elusive. Our study generates single-cell gene expression and T cell receptor profiles, along with validatory multimodal datasets from >100 high-grade serous ovarian cancer (HGSOC) samples, primarily from a phase II clinical trial (NCT04507841). Neoadjuvant monotherapy with the PARP inhibitor (PARPi) niraparib achieves impressive 62.5% and 73.6% response rates per RECIST v.1.1 and GCIG CA125, respectively. We identify effector regulatory T cells (eTregs) as key responders to HRD and neoadjuvant therapies, co-occurring with other tumor-reactive T cells, particularly terminally exhausted CD8+ T cells (Tex). TME-wide interferon signaling correlates with cancer cells upregulating MHC class II and co-inhibitory ligands, potentially driving Treg and Tex fates. Depleting eTregs in HRD mouse models, with or without PARP inhibition, significantly suppresses tumor growth without observable toxicities, underscoring the potential of eTreg-focused therapeutics for HGSOC and other HRD-related tumors.
RESUMO
Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8+ T cells deficient in both PD-1 and LAG-3, in contrast to CD8+ T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8+ T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8+ T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy.
Assuntos
Antígenos CD , Linfócitos T CD8-Positivos , Interferon gama , Proteína do Gene 3 de Ativação de Linfócitos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1 , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Animais , Interferon gama/metabolismo , Camundongos , Antígenos CD/metabolismo , Comunicação Autócrina , Humanos , Melanoma/imunologia , Melanoma/tratamento farmacológico , Feminino , Linhagem Celular Tumoral , Melanoma Experimental/imunologia , Exaustão das Células TRESUMO
MXRA8 is a receptor for chikungunya (CHIKV) and other arthritogenic alphaviruses with mammalian hosts. However, mammalian MXRA8 does not bind to alphaviruses that infect humans and have avian reservoirs. Here, we show that avian, but not mammalian, MXRA8 can act as a receptor for Sindbis, western equine encephalitis (WEEV), and related alphaviruses with avian reservoirs. Structural analysis of duck MXRA8 complexed with WEEV reveals an inverted binding mode compared with mammalian MXRA8 bound to CHIKV. Whereas both domains of mammalian MXRA8 bind CHIKV E1 and E2, only domain 1 of avian MXRA8 engages WEEV E1, and no appreciable contacts are made with WEEV E2. Using these results, we generated a chimeric avian-mammalian MXRA8 decoy-receptor that neutralizes infection of multiple alphaviruses from distinct antigenic groups in vitro and in vivo. Thus, different alphaviruses can bind MXRA8 encoded by different vertebrate classes with distinct engagement modes, which enables development of broad-spectrum inhibitors.
Assuntos
Alphavirus , Animais , Humanos , Febre de Chikungunya , Vírus Chikungunya/química , Mamíferos , Receptores Virais/metabolismoRESUMO
The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.
Assuntos
Limosilactobacillus reuteri , Melanoma , Microambiente Tumoral , Humanos , Dieta , Inibidores de Checkpoint Imunológico , Limosilactobacillus reuteri/metabolismo , Melanoma/terapia , Triptofano/metabolismo , Linfócitos T CD8-Positivos/imunologia , Receptores de Hidrocarboneto Arílico/agonistasRESUMO
Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5' end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown "Nuc-pocket" in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an "induce-and-lock" mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA , Antivirais/química , Nucleotídeos/química , Proteínas não Estruturais Virais/metabolismoRESUMO
The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue.
Assuntos
Química Click , Imagem Óptica , Animais , Encéfalo , Sistemas de Liberação de Medicamentos , Mamíferos , Camundongos , Imagem Óptica/métodosRESUMO
Two HIV fusion-inhibitory lipopeptides (LP-97 and LP-98) were designed with highly potent, long-acting antiviral activity. Monotherapy using a low dose of LP-98 sharply reduced viral loads and maintained long-term viral suppression in 21 SHIVSF162P3-infected rhesus macaques. We found that five treated monkeys achieved potential posttreatment control (PTC) efficacy and had lower viral DNA in deep lymph nodes, whereas monkeys with a stable viral rebound had higher viral DNA in superficial lymph nodes. The tissues of PTC monkeys exhibited significantly decreased quantitative viral outgrowth and fewer PD-1+ central memory CD4+ T cells, and CD8+ T cells contributed to virologic control efficacy. Moreover, LP-98 administrated as a pre-exposure prophylaxis (PrEP) provided complete protection against SHIVSF162P3 and SIVmac239 infections in 51 monkeys via intrarectal, intravaginal, or intravenous challenge. In conclusion, our lipopeptides exhibit high potential as an efficient HIV treatment or prevention strategy.
Assuntos
Inibidores da Fusão de HIV/administração & dosagem , Lipopeptídeos/administração & dosagem , Profilaxia Pré-Exposição/métodos , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Feminino , Células HEK293 , Humanos , Macaca mulatta , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Resposta Viral Sustentada , Células U937 , Carga Viral/efeitos dos fármacosRESUMO
The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 1012 cyclic peptides to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gßγ signaling in cells through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors.
Assuntos
Peptídeos , Receptores Acoplados a Proteínas G , GTP Fosfo-Hidrolases , Nucleotídeos de Guanina , Nucleotídeos , Peptídeos/química , Peptídeos Cíclicos/farmacologiaRESUMO
The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Clostridioides difficile/genética , Lipoproteínas/genéticaRESUMO
Development of γ-secretase inhibitors (GSIs) and modulators (GSMs) represents an attractive therapeutic opportunity for Alzheimer's disease (AD) and cancers. However, how these GSIs and GSMs target γ-secretase has remained largely unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human γ-secretase bound individually to two GSI clinical candidates, Semagacestat and Avagacestat, a transition state analog GSI L685,458, and a classic GSM E2012, at overall resolutions of 2.6-3.1 Å. Remarkably, each of the GSIs occupies the same general location on presenilin 1 (PS1) that accommodates the ß strand from amyloid precursor protein or Notch, interfering with substrate recruitment. L685,458 directly coordinates the two catalytic aspartate residues of PS1. E2012 binds to an allosteric site of γ-secretase on the extracellular side, potentially explaining its modulating activity. Structural analysis reveals a set of shared themes and variations for inhibitor and modulator recognition that will guide development of the next-generation substrate-selective inhibitors.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Preparações Farmacêuticas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/ultraestrutura , Azepinas/farmacologia , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Modelos Biológicos , Modelos Moleculares , Oxidiazóis/química , Oxidiazóis/farmacologia , Presenilina-1/química , Presenilina-1/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Neoantigens arise from mutations in cancer cells and are important targets of T cell-mediated anti-tumor immunity. Here, we report the first open-label, phase Ib clinical trial of a personalized neoantigen-based vaccine, NEO-PV-01, in combination with PD-1 blockade in patients with advanced melanoma, non-small cell lung cancer, or bladder cancer. This analysis of 82 patients demonstrated that the regimen was safe, with no treatment-related serious adverse events observed. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination in all of the patients. The vaccine-induced T cells had a cytotoxic phenotype and were capable of trafficking to the tumor and mediating cell killing. In addition, epitope spread to neoantigens not included in the vaccine was detected post-vaccination. These data support the safety and immunogenicity of this regimen in patients with advanced solid tumors (Clinicaltrials.gov: NCT02897765).
Assuntos
Vacinas Anticâncer/imunologia , Imunoterapia/métodos , Medicina de Precisão/métodos , Idoso , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Melanoma/tratamento farmacológico , Melanoma/imunologia , Pessoa de Meia-Idade , Mutação , Nivolumabe/uso terapêutico , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/imunologiaRESUMO
Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α6-ß6S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.
Assuntos
Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Aciltransferases/metabolismo , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-AtividadeRESUMO
Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6 Å in complex with D-glucose and 3.7 Å with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P. falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.
Assuntos
Proteínas de Transporte de Monossacarídeos/ultraestrutura , Plasmodium falciparum/metabolismo , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/ultraestrutura , Sequência de Aminoácidos , Animais , Antimaláricos , Transporte Biológico , Glucose/metabolismo , Humanos , Malária , Malária Falciparum/parasitologia , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Parasitos , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Açúcares/metabolismoRESUMO
The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Células A549 , Animais , Criança , Pré-Escolar , Feminino , Mutação com Ganho de Função/genética , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Células-Tronco Embrionárias Murinas , Mutação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais , Domínios de Homologia de src/genéticaRESUMO
Activating mutations in NRAS account for 20%-30% of melanoma, but despite decades of research and in contrast to BRAF, no effective anti-NRAS therapies have been forthcoming. Here, we identify a previously uncharacterized serine/threonine kinase STK19 as a novel NRAS activator. STK19 phosphorylates NRAS to enhance its binding to its downstream effectors and promotes oncogenic NRAS-mediated melanocyte malignant transformation. A recurrent D89N substitution in STK19 whose alterations were identified in 25% of human melanomas represents a gain-of-function mutation that interacts better with NRAS to enhance melanocyte transformation. STK19D89N knockin leads to skin hyperpigmentation and promotes NRASQ61R-driven melanomagenesis in vivo. Finally, we developed ZT-12-037-01 (1a) as a specific STK19-targeted inhibitor and showed that it effectively blocks oncogenic NRAS-driven melanocyte malignant transformation and melanoma growth in vitro and in vivo. Together, our findings provide a new and viable therapeutic strategy for melanomas harboring NRAS mutations.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Melanoma/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Feminino , Células HEK293 , Humanos , Melanócitos/metabolismo , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Mutação , Fosforilação , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genéticaRESUMO
Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable â¼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/ultraestrutura , Peptídeos/metabolismo , Venenos de Aranha/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetulus , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Células HEK293 , Humanos , Ativação do Canal Iônico , Peptídeos/toxicidade , Domínios Proteicos , Venenos de Aranha/toxicidade , Aranhas , Bloqueadores do Canal de Sódio Disparado por Voltagem , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Asma/terapia , Mastócitos/enzimologia , Mastócitos/imunologia , Triptases/antagonistas & inibidores , Triptases/imunologia , Adolescente , Regulação Alostérica/imunologia , Animais , Linhagem Celular , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , CoelhosRESUMO
The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.
Assuntos
Proteína 9 Associada à CRISPR/antagonistas & inibidores , Sistemas CRISPR-Cas/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , DNA/metabolismo , Endonucleases/metabolismo , Edição de Genes/métodos , Genoma , Bibliotecas de Moléculas Pequenas , Streptococcus pyogenes/genética , Especificidade por SubstratoRESUMO
Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.