RESUMEN
The symbiotic relationships shared between humans and their gastrointestinal parasites present opportunities to discover novel therapies for inflammatory diseases. A prime example of this phenomenon is the interaction of humans and roundworms such as the hookworm, Necator americanus. Epidemiological observations, animal studies and clinical trials using experimental human hookworm infection show that hookworms can suppress inflammation in a safe and well-tolerated way, and that the key to their immunomodulatory properties lies within their secreted proteome. Herein we describe the identification of 2 netrin domain-containing proteins from the N. americanus secretome, and explore their potential in treating intestinal inflammation in mouse models of ulcerative colitis. One of these proteins, subsequently named Na-AIP-1, was effective at suppressing disease when administered prophylactically in the acute TNBS-induced model of colitis. This protective effect was validated in the more robust CD4 T cell transfer model of chronic colitis, where prophylactic Na-AIP-1 reduced T-cell-dependent type-1 cytokine responses in the intestine and the associated intestinal pathology. Mechanistic studies revealed that depletion of CD11c+ cells abrogated the protective anticolitic effect of Na-AIP-1. Next generation sequencing of colon tissue in the T-cell transfer model of colitis revealed that Na-AIP-1 induced a transcriptomic profile associated with the downregulation of metabolic and signaling pathways involved in type-1 inflammation, notably TNF. Finally, co-culture of Na-AIP-1 with a human monocyte-derived M1 macrophage cell line resulted in significantly reduced secretion of TNF. Na-AIP-1 is now a candidate for clinical development as a novel therapeutic for the treatment of human inflammatory bowel diseases.
Asunto(s)
Antiinflamatorios/administración & dosificación , Linfocitos T CD4-Positivos/inmunología , Colitis Ulcerosa/prevención & control , Proteínas del Helminto/administración & dosificación , Necator americanus/química , Netrinas/administración & dosificación , Animales , Linfocitos T CD4-Positivos/trasplante , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/inmunología , Modelos Animales de Enfermedad , Femenino , Proteínas del Helminto/química , Proteínas del Helminto/genética , Infecciones por Uncinaria/metabolismo , Humanos , Masculino , Inhibidores de la Metaloproteinasa de la Matriz/química , Ratones Endogámicos C57BL , Ratones Noqueados , Netrinas/análisis , Proteínas Recombinantes/administración & dosificaciónRESUMEN
Inflammatory bowel diseases (IBDs) are a set of complex and debilitating diseases for which there is no satisfactory treatment. Recent studies have shown that small peptides show promise for reducing inflammation in models of IBD. However, these small peptides are likely to be unstable and rapidly cleared from the circulation, and therefore, if not modified for better stability, represent non-viable drug leads. We hypothesized that improving the stability of these peptides by grafting them into a stable cyclic peptide scaffold may enhance their therapeutic potential. Using this approach, we have designed a novel cyclic peptide that comprises a small bioactive peptide from the annexin A1 protein grafted into a sunflower trypsin inhibitor cyclic scaffold. We used native chemical ligation to synthesize the grafted cyclic peptide. This engineered cyclic peptide maintained the overall fold of the naturally occurring cyclic peptide, was more effective at reducing inflammation in a mouse model of acute colitis than the bioactive peptide alone, and showed enhanced stability in human serum. Our findings suggest that the use of cyclic peptides as structural backbones offers a promising approach for the treatment of IBD and potentially other chronic inflammatory conditions.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Colon/efectos de los fármacos , Modelos Animales de Enfermedad , Fármacos Gastrointestinales/uso terapéutico , Modelos Moleculares , Péptidos Cíclicos/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Colon/inmunología , Colon/patología , Diseño de Fármacos , Estabilidad de Medicamentos , Fármacos Gastrointestinales/síntesis química , Fármacos Gastrointestinales/química , Humanos , Masculino , Ratones Endogámicos C57BL , Tamaño de los Órganos/efectos de los fármacos , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Pliegue de Proteína , Estabilidad Proteica , Proteolisis , Distribución Aleatoria , Suero/enzimología , Organismos Libres de Patógenos EspecíficosRESUMEN
Granulins are a family of protein growth factors that are involved in cell proliferation. An orthologue of granulin from the human parasitic liver fluke Opisthorchis viverrini, known as Ov-GRN-1, induces angiogenesis and accelerates wound repair. Recombinant Ov-GRN-1 production is complex and poses an obstacle for clinical development. To identify the bioactive region(s) of Ov-GRN-1, four truncated N-terminal analogues were synthesized and characterized structurally using NMR spectroscopy. Peptides that contained only two native disulfide bonds lack the characteristic granulin ß-hairpin structure. Remarkably, the introduction of a non-native disulfide bond was critical for formation of ß-hairpin structure. Despite this structural difference, both two and three disulfide-bonded peptides drove proliferation of a human cholangiocyte cell line and demonstrated potent wound healing in mice. Peptides derived from Ov-GRN-1 are leads for novel wound healing therapeutics, as they are likely less immunogenic than the full-length protein and more convenient to produce.