RESUMEN
Toll-like receptor 7 (TLR7) and TLR8 are single-stranded RNA-sensing endosomal pattern recognition receptors that evolved to defend against viral infections. However, aberrant TLR7/8 activation by endogenous ligands has been implicated in the pathogenesis of autoimmune diseases including systemic lupus erythematosus. TLR activation and type I interferon (IFN) were shown recently to impart resistance to glucocorticoids (GC), which are part of the standard of care for multiple autoimmune diseases. While GCs are effective, a plethora of undesirable effects limit their use. New treatment approaches that allow for the use of lower and safer doses of GCs would be highly beneficial. Herein, we report that a dual TLR7/8 inhibitor (TLR7/8i) increases the effectiveness of GCs in inflammatory settings. Human peripheral blood mononuclear cell studies revealed increased GC sensitivity in the presence of TLR7/8i for reducing inflammatory cytokine production, a synergistic effect that was most pronounced in myeloid cells, particularly monocytes. Gene expression analysis by NanoString and single-cell RNA sequencing revealed that myeloid cells were substantially impacted by combining low-dose TLR7/8i and GC, as evidenced by the effects on nuclear factor-kappa B-regulated cytokines and GC-response genes, although IFNs were affected to a smaller degree. Low dose of TLR7/8i plus GC was more efficacious then either agent alone in the MRL/lpr mouse model of lupus, with improved proteinuria and survival. Overall, our findings indicate a GC-sparing potential for TLR7/8i compounds, suggesting TLR7/8i may offer a new strategy for the treatment of autoimmune diseases. SIGNIFICANCE STATEMENT: Some features of autoimmune diseases may be resistant to glucocorticoids, mediated at least in part by toll-like receptor (TLR) activation, necessitating higher doses that are associated with considerable toxicities. We demonstrate that TLR7/8 inhibition and glucocorticoids work synergistically to reduce inflammation in a cell-type specific manner and suppress disease in a mouse model of lupus. TLR7/8 inhibition is a promising strategy for the treatment of autoimmune diseases and has glucocorticoid-sparing potential.
Asunto(s)
Lupus Eritematoso Sistémico , Receptor Toll-Like 7 , Ratones , Animales , Humanos , Receptor Toll-Like 7/metabolismo , Glucocorticoides/farmacología , Leucocitos Mononucleares/metabolismo , Ratones Endogámicos MRL lpr , Receptores Toll-Like , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/genéticaRESUMEN
TL-895 (formerly known as M7583) is a potent, highly selective, adenosine triphosphate (ATP)-competitive, second-generation, irreversible inhibitor of Bruton's tyrosine kinase (BTK). We characterized its biochemical and cellular effects in in vitro and in vivo models. TL-895 was evaluated preclinically for potency against BTK using IC50 concentration-response curves; selectivity using a 270-kinase panel; BTK phosphorylation in Ramos Burkitt's lymphoma cells by ProteinSimple Wes analysis of one study; anti-proliferative effects in primary chronic lymphocytic leukemia (CLL) blasts; cell viability effects in diffuse large B-cell lymphoma (DLBCL) and mantle-cell lymphoma (MCL) cell lines; effects on antibody-dependent cell-mediated cytotoxicity (ADCC) from Daudi cells and chromium-51 release from human tumor cell lines; and efficacy in vivo using four MCL xenograft model and 21 DLBCL patient-derived xenograft (PDX) models (subtypes: 9 ABC, 11 GCB, 1 Unclassified). TL-895 was active against recombinant BTK (average IC50 1.5 nM) and inhibited only three additional kinases with IC50 within tenfold of BTK activity. TL-895 inhibited BTK auto-phosphorylation at the Y223 phosphorylation site (IC50 1-10 nM). TL-895 inhibited the proliferation of primary CLL blasts in vitro and inhibited growth in a subset of activated DLBCL and MCL cell lines. TL-895 inhibited the ADCC mechanism of therapeutic antibodies only at supra-clinical exposure levels. TL-895 significantly inhibited tumor growth in the Mino MCL xenograft model and in 5/21 DLBCL PDX models relative to vehicle controls. These findings demonstrate the potency of TL-895 for BTK and its efficacy in models of B-cell lymphoma despite its refined selectivity.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Linfoma de Células B Grandes Difuso , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Linfocitos B/metabolismo , Agammaglobulinemia Tirosina Quinasa , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Linfoma de Células B Grandes Difuso/patologíaRESUMEN
Toll-like receptor (TLR) 7 and TLR8 are transmembrane receptors that recognize single-stranded RNA. Activation of these receptors results in immune cell stimulation and inflammatory cytokine production, which is normally a protective host response. However, aberrant activation of TLR7/8 is potentially pathogenic and linked to progression of certain autoimmune diseases such as lupus. Thus, we hypothesize that an inhibitor that blocks TLR7/8 would be an effective therapeutic treatment. Prior efforts to develop inhibitors of TLR7/8 have been largely unsuccessful as a result of the challenge of producing a small-molecule inhibitor for these difficult targets. Here, we report the characterization of M5049 and compound 2, molecules which were discovered in a medicinal chemistry campaign to produce dual TLR7/8 inhibitors with drug-like properties. Both compounds showed potent and selective activity in a range of cellular assays for inhibition of TLR7/8 and block synthetic ligands and natural endogenous RNA ligands such as microRNA and Alu RNA. M5049 was found to be potent in vivo as TLR7/8 inhibition efficaciously treated disease in several murine lupus models and, interestingly, was efficacious in a disease context in which TLR7/8 activity has not previously been considered a primary disease driver. Furthermore, M5049 had greater potency in disease models than expected based on its in vitro potency and pharmacokinetic/pharmacodynamic properties. Because of its preferential accumulation in tissues, and ability to block multiple TLR7/8 RNA ligands, M5049 may be efficacious in treating autoimmunity and has the potential to provide benefit to a variety of patients with varying disease pathogenesis. SIGNIFICANCE STATEMENT: This study reports discovery of a novel toll-like receptor (TLR) 7 and TLR8 inhibitor (M5049); characterizes its binding mode, potency/selectivity, and pharmacokinetic and pharmacodynamic properties; and demonstrates its potential for treating autoimmune diseases in two mouse lupus models. TLR7/8 inhibition is unique in that it may block both innate and adaptive autoimmunity; thus, this study suggests that M5049 has the potential to benefit patients with autoimmune diseases.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Descubrimiento de Drogas , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 8/antagonistas & inhibidores , Animales , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Conformación Proteica , Receptor Toll-Like 7/química , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/química , Receptor Toll-Like 8/metabolismoRESUMEN
TLR7 and TLR8 are pattern recognition receptors that reside in the endosome and are activated by ssRNA molecules. TLR7 and TLR8 are normally part of the antiviral defense response, but they have also been implicated as drivers of autoimmune diseases such as lupus. The receptors have slightly different ligand-binding specificities and cellular expression patterns that suggest they have nonredundant specialized roles. How the roles of TLR7 and TLR8 differ may be determined by which cell types express each TLR and how the cells respond to activation of each receptor. To provide a better understanding of the effects of TLR7/8 activation, we have characterized changes induced by TLR-specific agonists in different human immune cell types and defined which responses are a direct consequence of TLR7 or TLR8 activation and which are secondary responses driven by type I IFN or cytokines produced subsequent to the primary response. Using cell sorting, gene expression analysis, and intracellular cytokine staining, we have found that the IFN regulatory factor (IRF) and NF-κB pathways are differentially activated downstream of the TLRs in various cell types. Studies with an anti-IFNAR Ab in human cells and lupus mice showed that inhibiting IFN activity can block secondary IFN-induced gene expression changes downstream of TLR7/8 activation, but not NF-κB-regulated genes induced directly by TLR7/8 activation at earlier timepoints. In summary, these results elucidate the different roles TLR7 and TLR8 play in immunity and inform strategies for potential treatment of autoimmune diseases driven by TLR7/8 activation.
Asunto(s)
Factores Reguladores del Interferón/metabolismo , Lupus Eritematoso Sistémico/inmunología , FN-kappa B/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Animales , Autoanticuerpos/sangre , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación , Interferón-alfa/farmacología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/genética , Ratones , Ratones Endogámicos DBA , Modelos Biológicos , Células Mieloides/clasificación , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistasRESUMEN
Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores Fc/metabolismo , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismo , Agammaglobulinemia Tirosina Quinasa , Línea Celular Tumoral , Análisis por Conglomerados , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Bruton's tyrosine kinase (Btk) is expressed in a variety of immune cells and previous work has demonstrated that blocking Btk is a promising strategy for treating autoimmune diseases. Herein, we utilized a tool Btk inhibitor, M7583, to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon. In BXSB-Yaa lupus mice, Btk inhibition reduced autoantibodies, nephritis, and mortality. In the pristane-induced DBA/1 lupus model, Btk inhibition suppressed arthritis, but autoantibodies and the IFN gene signature were not significantly affected; suggesting efficacy was mediated through inhibition of Fc receptors. In vitro studies using primary human macrophages revealed that Btk inhibition can block activation by immune complexes and TLR7 which contributes to tissue damage in SLE. Overall, our results provide translational insight into how Btk inhibition may provide benefit to a variety of SLE patients by affecting both BCR and FcR signaling.
Asunto(s)
Lupus Eritematoso Sistémico/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa , Animales , Artritis/tratamiento farmacológico , Artritis/patología , Autoanticuerpos/sangre , Modelos Animales de Enfermedad , Femenino , Articulaciones del Pie/efectos de los fármacos , Articulaciones del Pie/patología , Humanos , Inmunosupresores , Interferón Tipo I/inmunología , Riñón/efectos de los fármacos , Riñón/patología , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Nefritis/tratamiento farmacológico , Nefritis/patología , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Proteinuria/tratamiento farmacológico , Proteinuria/patología , Terpenos , Receptor Toll-Like 7/inmunologíaRESUMEN
In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de la Mielina/metabolismo , Vaina de Mielina/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/citología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/fisiología , Oligodendroglía , Nervio Óptico/citología , Ratas , Células Madre , Factores de Tiempo , Quinasas Asociadas a rho/metabolismoRESUMEN
Human immunodeficiency virus (HIV)-positive persons are predisposed to pulmonary infections, even after receiving effective highly active antiretroviral therapy. The reasons for this are unclear but may involve changes in innate immune function. HIV type 1 infection of macrophages impairs effector functions, including cytokine production. We observed decreased constitutive tumor necrosis factor alpha (TNF-alpha) concentrations and increased soluble tumor necrosis factor receptor type II (sTNFRII) in bronchoalveolar lavage fluid samples from HIV-positive subjects compared to healthy controls. Moreover, net proinflammatory TNF-alpha activity, as measured by the TNF-alpha/sTNFRII ratio, decreased as HIV-related disease progressed, as manifested by decreasing CD4 cell count and increasing HIV RNA (viral load). Since TNF-alpha is an important component of the innate immune system and is produced upon activation of Toll-like receptor (TLR) pathways, we hypothesized that the mechanism associated with deficient TNF-alpha production in the lung involved altered TLR expression or a deficit in the TLR signaling cascade. We found decreased Toll-like receptor 1 (TLR1) and TLR4 surface expression in HIV-infected U1 monocytic cells compared to the uninfected parental U937 cell line and decreased TLR message in alveolar macrophages (AMs) from HIV-positive subjects. In addition, stimulation with TLR1/2 ligand (Pam(3)Cys) or TLR4 ligand (lipopolysaccharide) resulted in decreased intracellular phosphorylated extracellular signal-regulated kinase and subsequent decreased transcription and expression of TNF-alpha in U1 cells compared to U937 cells. AMs from HIV-positive subjects also showed decreased TNF-alpha production in response to these TLR2 and TLR4 ligands. We postulate that HIV infection alters expression of TLRs with subsequent changes in mitogen-activated protein kinase signaling and cytokine production that ultimately leads to deficiencies of innate immune responses that predispose HIV-positive subjects to infection.