RESUMO
Exposure to nonself red blood cell (RBC) antigens, either from transfusion or pregnancy, may result in alloimmunization and incompatible RBC clearance. First described as a pregnancy complication 80 years ago, hemolytic disease of the fetus and newborn (HDFN) is caused by alloimmunization to paternally derived RBC antigens. Despite the morbidity/mortality of HDFN, women at risk for RBC alloimmunization have few therapeutic options. Given that alloantibodies to antigens in the KEL family are among the most clinically significant, we developed a murine model with RBC-specific expression of the human KEL antigen to evaluate the impact of maternal/fetal KEL incompatibility. After exposure to fetal KEL RBCs during successive pregnancies with KEL-positive males, 21 of 21 wild-type female mice developed anti-KEL alloantibodies; intrauterine fetal anemia and/or demise occurred in a subset of KEL-positive pups born to wild type, but not agammaglobulinemic mothers. Similar to previous observations in humans, pregnancy-associated alloantibodies were detrimental in a transfusion setting, and transfusion-associated alloantibodies were detrimental in a pregnancy setting. This is the first pregnancy-associated HDFN model described to date, which will serve as a platform to develop targeted therapies to prevent and/or mitigate the dangers of RBC alloantibodies to fetuses and newborns.
Assuntos
Anemia Hemolítica/imunologia , Eritrócitos/citologia , Isoanticorpos/imunologia , Sistema do Grupo Sanguíneo de Kell/imunologia , Modelos Animais , Anemia Hemolítica/genética , Animais , Transfusão de Sangue , Citocinas/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Imunoglobulina G/imunologia , Sistema do Grupo Sanguíneo de Kell/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , PrenhezRESUMO
BACKGROUND: Red blood cell (RBC) alloantibodies to nonself antigens may develop after transfusion or pregnancy, leading to morbidity and mortality in the form of hemolytic transfusion reactions or hemolytic disease of the newborn. A better understanding of the mechanisms of RBC alloantibody induction, or strategies to mitigate the consequences of such antibodies, may ultimately improve transfusion safety. However, such studies are inherently difficult in humans. STUDY DESIGN AND METHODS: We recently generated transgenic mice with RBC-specific expression of the human KEL glycoprotein, specifically the KEL2 or KEL1 antigens. Herein, we investigate recipient alloimmune responses to transfused RBCs in this system. RESULTS: Transfusion of RBCs from KEL2 donors into wild-type recipients (lacking the human KEL protein but expressing the murine KEL ortholog) resulted in dose-dependent anti-KEL glycoprotein immunoglobulin (Ig)M and IgG antibody responses, enhanced by recipient inflammation with poly(I:C). Boostable responses were evident upon repeat transfusion, with morbid-appearing alloimmunized recipients experiencing rapid clearance of transfused KEL2 but not control RBCs. Although KEL1 RBCs were also immunogenic after transfusion into wild-type recipients, transfusion of KEL1 RBCs into KEL2 recipients or vice versa failed to lead to detectable anti-KEL1 or anti-KEL2 responses. CONCLUSIONS: This murine model, with reproducible and clinically significant KEL glycoprotein alloantibody responses, provides a platform for future mechanistic studies of RBC alloantibody induction and consequences. Long-term translational goals of these studies include improving transfusion safety for at-risk patients.
Assuntos
Transfusão de Eritrócitos/métodos , Eritrócitos/imunologia , Isoanticorpos/biossíntese , Sistema do Grupo Sanguíneo de Kell/imunologia , Anemia Hemolítica/genética , Anemia Hemolítica/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/genética , Incompatibilidade de Grupos Sanguíneos/genética , Incompatibilidade de Grupos Sanguíneos/imunologia , Eritrócitos/metabolismo , Humanos , Inflamação/imunologia , Sistema do Grupo Sanguíneo de Kell/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Poli I-CRESUMO
We herein report the discovery, synthesis, and evolution of a series of indazoles and azaindazoles as CNS-penetrant IRAK4 inhibitors. Described is the use of structure-based and property-based drug design strategically leveraged to guide the property profile of a key series into a favorable property space while maintaining potency and selectivity. Our rationale that led toward functionalities with potency improvements, CNS-penetration, solubility, and favorable drug-like properties is portrayed. In vivo evaluation of an advanced analogue showed significant, dose-dependent modulation of inflammatory cytokines in a mouse model. In pursuit of incorporating a highly engineered bridged ether that was crucial to metabolic stability in this series, significant synthetic challenges were overcome to enable the preparation of the analogues.
RESUMO
Interleukin receptor-associated kinase 4 (IRAK4) is a key node of signaling within the innate immune system that regulates the production of inflammatory cytokines and chemokines. The presence of damage-associated molecular patterns (DAMPs) after tissue damage such as stroke or traumatic brain injury (TBI) initiates signaling through the IRAK4 pathway that can lead to a feed-forward inflammatory loop that can ultimately hinder patient recovery. Herein, we describe the first potent, selective, and CNS-penetrant IRAK4 inhibitors for the treatment of neuroinflammation. Lead compounds from the series were evaluated in CNS PK/PD models of inflammation, as well as a mouse model of ischemic stroke. The SAR optimization detailed within culminates in the discovery of BIO-7488, a highly selective and potent IRAK4 inhibitor that is CNS penetrant and has excellent ADME properties.
Assuntos
Quinases Associadas a Receptores de Interleucina-1 , AVC Isquêmico , Camundongos , Animais , Humanos , Transdução de Sinais , Citocinas , Pirimidinas/farmacologia , Pirimidinas/uso terapêuticoRESUMO
Interleukin receptor associated kinase 4 (IRAK4) plays an important role in innate immune signaling through Toll-like and interleukin-1 receptors and represents an attractive target for the treatment of inflammatory diseases and cancer. We previously reported the development of a potent, selective, and brain-penetrant imidazopyrimidine series of IRAK4 inhibitors. However, lead molecule BIO-7488 (1) suffered from low solubility which led to variable PK, compound accumulation, and poor in vivo tolerability. Herein, we describe the discovery of a series of pyridone analogs with improved solubility which are highly potent, selective and demonstrate desirable PK profiles including good oral bioavailability and excellent brain penetration. BIO-8169 (2) reduced the in vivo production of pro-inflammatory cytokines, was well tolerated in safety studies in rodents and dog at margins well above the predicted efficacious exposure and showed promising results in a mouse model for multiple sclerosis.
Assuntos
Encéfalo , Quinases Associadas a Receptores de Interleucina-1 , Inibidores de Proteínas Quinases , Animais , Cães , Masculino , Camundongos , Ratos , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Descoberta de Drogas , Encefalomielite Autoimune Experimental/tratamento farmacológico , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Pirimidinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/síntese química , Pirimidinas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
BACKGROUND: KEL1, also known as "K", is one of the most immunogenic red blood cell (RBC) antigens. KEL2, also known as "k," differs from KEL1 by a single amino acid. Anti-Kell system antibodies can lead to significant adverse clinical outcomes in humans, including hemolytic complications in alloimmunized transfusion recipients or in infants of alloimmunized mothers. To provide a platform for in-depth immunologic studies of alloimmunization and subsequent sequelae, we generated transgenic mice expressing the human KEL1 or KEL2 antigens. STUDY DESIGN AND METHODS: Vectors were created in which cDNAs encoding either KEL1 or KEL2 were regulated by an erythroid specific ß-globin promoter and enhancer. Pronuclear microinjections were carried out into a C57BL6 background, and founder pups were identified by polymerase chain reaction and screened for expression by flow cytometry. RBC life span and antigen stability were assessed by dye labeling RBCs, transfusing into agammaglobulinemic (µMT) recipients, and tracking by flow cytometry. RESULTS: The expression of either KEL1 or KEL2 is RBC specific and first occurs on early RBC precursors. Both KEL1 and KEL2 RBCs have a normal circulatory life span and stable antigen expression. Expression of KEL1 or KEL2 does not result in altered levels of murine Kell, and resulting RBCs have normal hematologic variables. CONCLUSION: The KEL1 and KEL2 mice represent the first murine system of RBC immunity with antithetical antigens, allowing a more precise modeling of human RBC immunology in general and also a platform for development of novel therapeutics to prevent or minimize the dangers of RBC alloimmunization to the KEL1 and KEL2 antigens in particular.
Assuntos
Eritrócitos/imunologia , Sistema do Grupo Sanguíneo de Kell/genética , Sistema do Grupo Sanguíneo de Kell/imunologia , Camundongos Transgênicos , Modelos Animais , Animais , Transfusão de Sangue , Epitopos/genética , Epitopos/imunologia , Eritrócitos/citologia , Biblioteca Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transgenes/genéticaRESUMO
The main complication of replacement therapy with factor in hemophilia A (HemA) is the formation of inhibitors (neutralizing anti-factor VIII [FVIII] antibodies) in â¼30% of severe HemA patients. Because these inhibitors render replacement FVIII treatment essentially ineffective, preventing or eliminating them is of top priority in disease management. The extended half-life recombinant FVIII Fc fusion protein (rFVIIIFc) is an approved therapy for HemA patients. In addition, it has been reported that rFVIIIFc may induce tolerance to FVIII more readily than FVIII alone in HemA patients that have developed inhibitors. Given that the immunoglobulin G1 Fc region has the potential to interact with immune cells expressing Fc receptors (FcRs) and thereby affect the immune response to rFVIII, we investigated how human macrophages, expressing both FcRs and receptors reported to bind FVIII, respond to rFVIIIFc. We show herein that rFVIIIFc, but not rFVIII, uniquely skews macrophages toward an alternatively activated regulatory phenotype. rFVIIIFc initiates signaling events that result in morphological changes, as well as a specific gene expression and metabolic profile that is characteristic of the regulatory type Mox/M2-like macrophages. Further, these changes are dependent on rFVIIIFc-FcR interactions. Our findings elucidate mechanisms of potential immunomodulatory properties of rFVIIIFc.
Assuntos
Fator VIII/farmacologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Células Cultivadas , Fator VIII/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Hemofilia A/tratamento farmacológico , Hemofilia A/patologia , Humanos , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Leucócitos Mononucleares/citologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.