RESUMEN
Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Glucosamina/farmacología , Glucolípidos/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/toxicidad , Animales , Femenino , Glucosamina/síntesis química , Glucosamina/metabolismo , Glucosamina/toxicidad , Glucolípidos/síntesis química , Glucolípidos/metabolismo , Glucolípidos/toxicidad , Humanos , Inflamasomas/metabolismo , Interleucina-1/metabolismo , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
Cervical carcinomas express human papillomavirus (HPV) E6 and E7 oncoproteins, which are required to maintain the proliferative state of cancer cells. Repression of E6 and E7 expression results in acquisition of senescent phenotype and in the rescue of functional p53 and p105(Rb) pathways; therefore, therapies directed against either viral protein may be beneficial. However, the systems to study HPV in vitro are technically difficult and not convenient for screening of antiviral compounds. This has hampered the discovery of drugs against HPV. Here we describe the generation and use of a high-throughput screening system based on keratinocytes stably transfected with a reporter construct containing the regulatory sequence of E6 and E7 gene transcription (LCR) that allows easy detection of inhibitors of E6 and E7 expression in libraries of synthetic or biological compounds. The assay was used to screen a wide panel of cytokines: among them, IL-4, IL-13, TGF-beta1, TGF-beta2, TGF-beta3, TNF-alpha, IFN-alpha, and IFN-beta were found to induce a strong inhibition of the LCR activity. Our assay provides a validated tool in the search for drugs against HPV-associated cervical carcinomas and allowed the first systematic analysis of the effect of cytokines on the HPV-16 LCR transcriptional activity.