RESUMEN
Psoriasis vulgaris (PV) results from activation of IL-23/Th17 immune pathway and is further amplified by cytokines/chemokines from skin cells. Among skin-derived pro-inflammatory cytokines, IL-36 family members are highly upregulated in PV patients and play a critical role in general pustular psoriasis. However, there is limited data showing crosstalk between the IL-23 and IL-36 pathways in PV. Herein, potential attenuation of skin inflammation in the IL-23-induced mouse model of psoriasiform dermatitis by functional inhibition of IL-36 receptor (IL-36R) was interrogated. Anti-mouse IL-36R monoclonal antibodies (mAbs) were generated and validated in vitro by inhibiting IL-36α-induced secretion of CXCL1 from NIH 3T3 cells. Antibody target engagement was demonstrated by inhibition of CXCL1 production in a novel acute model of IL-36α systemic injection in mice. In addition, anti-IL-36R mAbs inhibited tissue inflammation and inflammatory gene expression in an IL-36α ear injection model of psoriasiform dermatitis demonstrating engagement of the target in the ear skin. To elucidate the possible role of IL-36 signalling in IL-23/Th17 pathway, the ability of anti-IL-36R mAbs to inhibit skin inflammation in an IL-23 ear injection model was assessed. Inhibiting the IL-36 pathway resulted in significant attenuation of skin thickening and psoriasis-relevant gene expression. Taken together, these data suggest a role for IL-36 signalling in the IL-23/Th17 signalling axis in PV.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Dermatitis/inmunología , Inflamación/inmunología , Interleucinas/inmunología , Psoriasis/inmunología , Receptores de Interleucina/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/uso terapéutico , Quimiocina CXCL1/metabolismo , Citocinas/metabolismo , Dermatitis/terapia , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Inflamación/metabolismo , Interleucina-1/inmunología , Interleucina-23/farmacología , Ligandos , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Psoriasis/terapia , Ratas , Ratas Sprague-Dawley , Receptores de Interleucina/inmunología , Receptores de Interleucina-1/inmunología , Transducción de Señal , Piel/metabolismo , Piel/patología , Células Th17/citologíaRESUMEN
Ligand-binding assays are the linchpin of drug discovery and medicinal chemistry. Cell-surface receptors and their ligands have traditionally been characterized by radioligand-binding assays, which have low temporal and spatial resolution and entail safety risks. Here, we report a powerful alternative (GlycoFRET), where terbium-labeled fluorescent reporters are irreversibly attached to receptors by metabolic glycan engineering. For the first time, we show time-resolved fluorescence resonance energy transfer between receptor glycans and fluorescently labeled ligands. We describe GlycoFRET for a GPI-anchored receptor, a G-protein-coupled receptor, and a heterodimeric cytokine receptor in living cells with excellent sensitivity and high signal-to-background ratios. In contrast to previously described methods, GlycoFRET does not require genetic engineering or antibodies to label receptors. Given that all cell-surface receptors are glycosylated, we expect that GlycoFRET can be generalized with applications in chemical biology and biotechnology, such as target engagement, receptor pharmacology, and high-throughput screening.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Ligadas a GPI/metabolismo , Polisacáridos/metabolismo , Receptores de Superficie Celular/metabolismo , Supervivencia Celular , Receptores de Folato Anclados a GPI/metabolismo , Humanos , Ligandos , Receptores Histamínicos H3/metabolismo , Receptores de Interleucina/metabolismo , TerbioRESUMEN
IL-36 cytokines are pro-inflammatory members of the IL-1 family that are upregulated in inflammatory disorders. Specifically, IL-36γ is highly expressed in active psoriatic lesions and can drive pro-inflammatory processes in 3D human skin equivalents supporting a role for this target in skin inflammation. Small molecule antagonists of interleukins have been historically challenging to generate. Nevertheless, we performed a small molecule high-throughput screen to identify IL-36 antagonists using a novel TR-FRET binding assay. Several compounds, including 2-oxypyrimidine containing structural analogs of the marketed endothelin receptor A antagonist Ambrisentan, were identified as hits from the screen. A-552 was identified as a the most potent antagonist of human IL-36γ, but not the closely related family member IL-36α, was capable of attenuating IL-36γ induced responses in mouse and human disease models. Additionally, x-ray crystallography studies identified key amino acid residues in the binding pocket present in human IL-36γ that are absent in human IL-36α. A-552 represents a first-in-class small molecule antagonist of IL-36 signaling that could be used as a chemical tool to further investigate the role of this pathway in inflammatory skin diseases such as psoriasis.
Asunto(s)
Interleucina-1/antagonistas & inhibidores , Psoriasis/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Psoriasis/metabolismo , Psoriasis/patología , Piel/efectos de los fármacos , Piel/patología , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain. ppk1 null mutant larvae had normal external touch sensation and md neuron morphology but displayed striking alterations in crawling behavior. Loss of PPK1 function caused an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion resulted from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype was rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the PPK1 channel plays an essential role in controlling rhythmic locomotion and provide a powerful genetic model system for further analysis of central and peripheral control mechanisms and their role in movement disorders.