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1.
PLoS Pathog ; 20(8): e1012436, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39196893

RESUMEN

Viruses capable of causing persistent infection have developed sophisticated mechanisms for evading host immunity, and understanding these processes can reveal novel features of the host immune system. One such virus, human pegivirus (HPgV), infects ~15% of the global human population, but little is known about its biology beyond the fact that it does not cause overt disease. We passaged a pegivirus isolate of feral brown rats (RPgV) in immunodeficient laboratory mice to develop a mouse-adapted virus (maPgV) that established persistent high-titer infection in a majority of wild-type laboratory mice. maRPgV viremia was detected in the blood of mice for >300 days without apparent disease, closely recapitulating the hallmarks of HPgV infection in humans. We found a pro-viral role for type-I interferon in chronic infection; a lack of PD-1-mediated tolerance to PgV infection; and multiple mechanisms by which PgV immunity can be achieved by an immunocompetent host. These data indicate that the PgV immune evasion strategy has aspects that are both common and unique among persistent viral infections. The creation of maPgV represents the first PgV infection model in wild-type mice, thus opening the entire toolkit of the mouse host to enable further investigation of this persistent RNA virus infections.


Asunto(s)
Infecciones por Flaviviridae , Flaviviridae , Animales , Ratones , Infecciones por Flaviviridae/virología , Infecciones por Flaviviridae/inmunología , Flaviviridae/genética , Flaviviridae/inmunología , Infección Persistente/inmunología , Infección Persistente/virología , Ratas , Evasión Inmune , Ratones Endogámicos C57BL , Humanos
2.
Sci Transl Med ; 16(754): eadi6887, 2024 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-38959328

RESUMEN

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.


Asunto(s)
COVID-19 , Fosfatidilinositol 3-Quinasa Clase Ib , Inflamación , SARS-CoV-2 , Animales , Humanos , Ratones , Permeabilidad Capilar/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , COVID-19/patología , Tratamiento Farmacológico de COVID-19 , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Inflamación/patología , Pulmón/patología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones Endogámicos C57BL , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , SARS-CoV-2/fisiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/patología
3.
Science ; 384(6702): eade8520, 2024 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-38900864

RESUMEN

Unleashing antitumor T cell activity by checkpoint inhibitor immunotherapy is effective in cancer patients, but clinical responses are limited. Cytokine signaling through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway correlates with checkpoint immunotherapy resistance. We report a phase I clinical trial of the JAK inhibitor ruxolitinib with anti-PD-1 antibody nivolumab in Hodgkin lymphoma patients relapsed or refractory following checkpoint inhibitor immunotherapy. The combination yielded a best overall response rate of 53% (10/19). Ruxolitinib significantly reduced neutrophil-to-lymphocyte ratios and percentages of myeloid suppressor cells but increased numbers of cytokine-producing T cells. Ruxolitinib rescued the function of exhausted T cells and enhanced the efficacy of immune checkpoint blockade in preclinical solid tumor and lymphoma models. This synergy was characterized by a switch from suppressive to immunostimulatory myeloid cells, which enhanced T cell division.


Asunto(s)
Enfermedad de Hodgkin , Inhibidores de Puntos de Control Inmunológico , Inhibidores de las Cinasas Janus , Nitrilos , Nivolumab , Pirazoles , Pirimidinas , Linfocitos T , Adulto , Anciano , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Sinergismo Farmacológico , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/terapia , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia , Inhibidores de las Cinasas Janus/uso terapéutico , Quinasas Janus/metabolismo , Quinasas Janus/antagonistas & inhibidores , Nitrilos/uso terapéutico , Nivolumab/uso terapéutico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Linfocitos T/inmunología , Ratones Endogámicos C57BL , Ratones Endogámicos BALB C
4.
Nat Biomed Eng ; 8(5): 499-512, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38693431

RESUMEN

Bispecific T-cell engagers (BiTEs) bring together tumour cells and cytotoxic T cells by binding to specific cell-surface tumour antigens and T-cell receptors, and have been clinically successful for the treatment of B-cell malignancies. Here we show that a BiTE-sialidase fusion protein enhances the susceptibility of solid tumours to BiTE-mediated cytolysis of tumour cells via targeted desialylation-that is, the removal of terminal sialic acid residues on glycans-at the BiTE-induced T-cell-tumour-cell interface. In xenograft and syngeneic mouse models of leukaemia and of melanoma and breast cancer, and compared with the parental BiTE molecules, targeted desialylation via the BiTE-sialidase fusion proteins enhanced the formation of immunological synapses, T-cell activation and T-cell-mediated tumour-cell cytolysis in the presence of the target antigen. The targeted desialylation of tumour cells may enhance the potency of therapies relying on T-cell engagers.


Asunto(s)
Neuraminidasa , Animales , Neuraminidasa/metabolismo , Humanos , Ratones , Línea Celular Tumoral , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunología , Femenino , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Activación de Linfocitos , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/química , Ensayos Antitumor por Modelo de Xenoinjerto , Linfocitos T Citotóxicos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología
5.
PLoS Pathog ; 20(3): e1012095, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38512979

RESUMEN

The 1858C>T allele of the tyrosine phosphatase PTPN22 is present in 5-10% of the North American population and is strongly associated with numerous autoimmune diseases. Although research has been done to define how this allele potentiates autoimmunity, the influence PTPN22 and its pro-autoimmune allele has in anti-viral immunity remains poorly defined. Here, we use single cell RNA-sequencing and functional studies to interrogate the impact of this pro-autoimmune allele on anti-viral immunity during Lymphocytic Choriomeningitis Virus clone 13 (LCMV-cl13) infection. Mice homozygous for this allele (PEP-619WW) clear the LCMV-cl13 virus whereas wildtype (PEP-WT) mice cannot. This is associated with enhanced anti-viral CD4 T cell responses and a more immunostimulatory CD8α- cDC phenotype. Adoptive transfer studies demonstrated that PEP-619WW enhanced anti-viral CD4 T cell function through virus-specific CD4 T cell intrinsic and extrinsic mechanisms. Taken together, our data show that the pro-autoimmune allele of Ptpn22 drives a beneficial anti-viral immune response thereby preventing what is normally a chronic virus infection.


Asunto(s)
Enfermedades Autoinmunes , Coriomeningitis Linfocítica , Animales , Ratones , Alelos , Enfermedades Autoinmunes/genética , Autoinmunidad/genética , Monoéster Fosfórico Hidrolasas/genética , Tirosina
7.
Nat Chem Biol ; 20(8): 1000-1011, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38191941

RESUMEN

SLC15A4 is an endolysosome-resident transporter linked with autoinflammation and autoimmunity. Specifically, SLC15A4 is critical for Toll-like receptors (TLRs) 7-9 as well as nucleotide-binding oligomerization domain-containing protein (NOD) signaling in several immune cell subsets. Notably, SLC15A4 is essential for the development of systemic lupus erythematosus in murine models and is associated with autoimmune conditions in humans. Despite its therapeutic potential, the availability of quality chemical probes targeting SLC15A4 functions is limited. In this study, we used an integrated chemical proteomics approach to develop a suite of chemical tools, including first-in-class functional inhibitors, for SLC15A4. We demonstrate that these inhibitors suppress SLC15A4-mediated endolysosomal TLR and NOD functions in a variety of human and mouse immune cells; we provide evidence of their ability to suppress inflammation in vivo and in clinical settings; and we provide insights into their mechanism of action. Our findings establish SLC15A4 as a druggable target for the treatment of autoimmune and autoinflammatory conditions.


Asunto(s)
Proteómica , Animales , Humanos , Ratones , Proteómica/métodos , Antiinflamatorios/farmacología , Antiinflamatorios/química , Proteínas de Transporte de Nucleósidos/metabolismo , Proteínas de Transporte de Nucleósidos/antagonistas & inhibidores , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Ratones Endogámicos C57BL , Femenino , Proteínas del Tejido Nervioso , Proteínas de Transporte de Membrana
8.
Med ; 5(1): 42-61.e23, 2024 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-38181791

RESUMEN

BACKGROUND: Oral antiviral drugs with improved antiviral potency and safety are needed to address current challenges in clinical practice for treatment of COVID-19, including the risks of rebound, drug-drug interactions, and emerging resistance. METHODS: Olgotrelvir (STI-1558) is designed as a next-generation antiviral targeting the SARS-CoV-2 main protease (Mpro), an essential enzyme for SARS-CoV-2 replication, and human cathepsin L (CTSL), a key enzyme for SARS-CoV-2 entry into host cells. FINDINGS: Olgotrelvir is a highly bioavailable oral prodrug that is converted in plasma to its active form, AC1115. The dual mechanism of action of olgotrelvir and AC1115 was confirmed by enzyme activity inhibition assays and co-crystal structures of AC1115 with SARS-CoV-2 Mpro and human CTSL. AC1115 displayed antiviral activity by inhibiting replication of all tested SARS-CoV-2 variants in cell culture systems. Olgotrelvir also inhibited viral entry into cells using SARS-CoV-2 Spike-mediated pseudotypes by inhibition of host CTSL. In the K18-hACE2 transgenic mouse model of SARS-CoV-2-mediated disease, olgotrelvir significantly reduced the virus load in the lungs, prevented body weight loss, and reduced cytokine release and lung pathologies. Olgotrelvir demonstrated potent activity against the nirmatrelvir-resistant Mpro E166 mutants. Olgotrelvir showed enhanced oral bioavailability in animal models and in humans with significant plasma exposure without ritonavir. In phase I studies (ClinicalTrials.gov: NCT05364840 and NCT05523739), olgotrelvir demonstrated a favorable safety profile and antiviral activity. CONCLUSIONS: Olgotrelvir is an oral inhibitor targeting Mpro and CTSL with high antiviral activity and plasma exposure and is a standalone treatment candidate for COVID-19. FUNDING: Funded by Sorrento Therapeutics.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , COVID-19 , Inhibidores de Proteasa de Coronavirus , SARS-CoV-2 , Animales , Humanos , Ratones , Antivirales/farmacología , Antivirales/uso terapéutico , Catepsina L/antagonistas & inhibidores , COVID-19/prevención & control , Modelos Animales de Enfermedad , Ratones Transgénicos , Inhibidores de Proteasa de Coronavirus/química , Inhibidores de Proteasa de Coronavirus/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Tratamiento Farmacológico de COVID-19/métodos
9.
ACS Chem Biol ; 19(2): 254-265, 2024 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-38198472

RESUMEN

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines, including IL-1ß and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remain poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit the inflammasome assembly and activity. From this screen, we identify and profile inflammasome inhibition of 20 new covalent compounds across nine different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks the activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular cross-linking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, further support the continued development of electrophilic compounds that target reactive cysteine residues on NLRP3 to regulate its activation and activity.


Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal , Citocinas , Interleucina-1beta/metabolismo
10.
Proc Natl Acad Sci U S A ; 120(39): e2303455120, 2023 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-37722054

RESUMEN

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.


Asunto(s)
COVID-19 , SARS-CoV-2 , Femenino , Animales , Bovinos , Anticuerpos , Fragmentos Fab de Inmunoglobulinas/genética , Disulfuros
11.
Sci Signal ; 16(798): eabk3516, 2023 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-37582161

RESUMEN

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing antibody that neutralizes one virus but not a related virus. Through a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1-neutralizing antibody, to bind to the receptor binding domain of SARS-CoV-2 with >1000-fold increased affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of engineered CR3022 elucidated the molecular mechanisms by which the antibody can accommodate sequence differences in the epitopes between SARS-CoV-1 and SARS-CoV-2. This workflow provides a blueprint for the rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.


Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , SARS-CoV-2/genética , COVID-19/prevención & control , Anticuerpos Antivirales , Pruebas de Neutralización , Anticuerpos Neutralizantes
12.
Cell Rep ; 42(8): 112968, 2023 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-37578862

RESUMEN

The miR-17∼92 family microRNAs (miRNAs) play a key role in germinal center (GC) reaction through promoting T follicular helper (TFH) cell differentiation. It remains unclear whether they also have intrinsic functions in B cell differentiation and function. Here we show that mice with B cell-specific deletion of the miR-17∼92 family exhibit impaired GC reaction, plasma cell differentiation, and antibody production in response to protein antigen immunization and chronic viral infection. Employing CRISPR-mediated functional screening, we identify Socs3 as a key functional target of miR-17∼92 in regulating plasma cell differentiation. Mechanistically, SOCS3, whose expression is elevated in miR-17∼92 family-deficient B cells, interacts with NIK and promotes its ubiquitination and degradation, thereby impairing NF-κB signaling and plasma cell differentiation. This moderate increase in SOCS3 expression has little effect on IL-21-STAT3 signaling. Our study demonstrates differential sensitivity of two key signaling pathways to alterations in the protein level of an miRNA target gene.


Asunto(s)
MicroARNs , Ratones , Animales , MicroARNs/genética , MicroARNs/metabolismo , Linfocitos T Colaboradores-Inductores , Linfocitos B , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Diferenciación Celular/genética , Centro Germinal
13.
bioRxiv ; 2023 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-37398499

RESUMEN

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines including IL-1ß and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remains poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit inflammasome assembly and activity. From this screen we identify and profile inflammasome inhibition of 20 new covalent compounds across 9 different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular crosslinking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, suggests that NLRP3 serves as a cellular electrophile sensor important for coordinating inflammatory signaling in response to redox stress. Further, our results support the potential for covalent cysteine modification of NLRP3 for regulating inflammasome activation and activity.

14.
Mol Cell ; 83(10): 1725-1742.e12, 2023 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-37084731

RESUMEN

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.


Asunto(s)
Proteómica , Factores de Transcripción , Humanos , Proteómica/métodos , Cisteína/metabolismo , Ligandos
15.
bioRxiv ; 2023 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-36798389

RESUMEN

The histone methyltransferase enhancer of zeste homolog 2 (EZH2)-mediated epigenetic regulation of T cell differentiation in acute infection has been extensively investigated. However, the role of EZH2 in T cell exhaustion remains under-explored. Here, using in vitro exhaustion models, we demonstrated that transient inhibition of EZH2 in T cells before the phenotypic onset of exhaustion with a clinically approved inhibitor, Tazemetastat, delayed their dysfunctional progression and maintained T cell stemness and polyfunctionality while having no negative impact on cell proliferation. Tazemetestat induced T cell epigenetic reprogramming and increased the expression of the self-renewing T cell transcription factor TCF1 by reducing its promoter H3K27 methylation preferentially in rapidly dividing T cells. In a murine melanoma model, T cells pre-treated with tazemetastat exhibited a superior response to anti-PD-1 blockade therapy after adoptive transfer. Collectively, these data unveil the potential of transient epigenetic reprogramming as a potential intervention to be combined with checkpoint blockade for immune therapy.

16.
Front Immunol ; 13: 906355, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36189299

RESUMEN

Cytotoxic CD8 T cells are crucial for the host antigen-specific immune response to viral pathogens. Here we report the identification of an essential role for the serine/arginine-rich splicing factor (SRSF) 1 in CD8 T cell homeostasis and function. Specifically, SRSF1 is necessary for the maintenance of normal CD8 T lymphocyte numbers in the lymphoid compartment, and for the proliferative capacity and cytotoxic function of CD8 T cells. Furthermore, SRSF1 is required for antigen-specific IFN-γ cytokine responses in a viral infection challenge in mice. Transcriptomics analyses of Srsf1-deficient T cells reveal that SRSF1 controls proliferation, MAP kinase signaling and IFN signaling pathways. Mechanistically, SRSF1 controls the expression and activity of the Mnk2/p38-MAPK axis at the molecular level. Our findings reveal previously unrecognized roles for SRSF1 in the physiology and function of cytotoxic CD8 T lymphocytes and a potential molecular mechanism in viral immunopathogenesis.


Asunto(s)
Linfocitos T CD8-positivos , Citocinas , Factores de Empalme Serina-Arginina/inmunología , Animales , Arginina , Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Inmunidad , Ratones , Factores de Empalme de ARN , Serina , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo
17.
Nat Chem Biol ; 18(12): 1388-1398, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36097295

RESUMEN

The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.


Asunto(s)
Cisteína , Proteómica , Transducción de Señal , Citocinas , Isoformas de Proteínas
19.
iScience ; 25(9): 104914, 2022 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-35971553

RESUMEN

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we used a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduces the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity-matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for antiviral indications would benefit from affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

20.
Med ; 3(10): 705-721.e11, 2022 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-36044897

RESUMEN

BACKGROUND: The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant and its BA.X lineages, has rendered ineffective a number of previously FDA emergency use authorized SARS-CoV-2 neutralizing antibody therapies. Furthermore, those approved antibodies with neutralizing activity against Omicron BA.1 are reportedly ineffective against the subset of Omicron subvariants that contain a R346K substitution, BA.1.1, and the more recently emergent BA.2, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. METHODS: Following a campaign of antibody discovery based on the vaccination of Harbor H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. FINDINGS: STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against each of the tested Omicron subvariants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. CONCLUSIONS: With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for intravenous or intranasal use in human clinical trials. FUNDING: Funded by CRIPT (no. 75N93021R00014), DARPA (HR0011-19-2-0020), and NCI Seronet (U54CA260560).


Asunto(s)
Anticuerpos Neutralizantes , Tratamiento Farmacológico de COVID-19 , Administración Intranasal , Animales , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Humanos , Inmunoglobulina G , Glicoproteínas de Membrana , Ratones , Pruebas de Neutralización , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas del Envoltorio Viral
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