RESUMEN
We disclose here a panel of small-molecule TLR4 agonists (the FP20 series) whose structure is derived from previously developed TLR4 ligands (FP18 series). The new molecules have increased chemical stability and a shorter, more efficient, and scalable synthesis. The FP20 series showed selective activity as TLR4 agonists with a potency similar to FP18. Interestingly, despite the chemical similarity with the FP18 series, FP20 showed a different mechanism of action and immunofluorescence microscopy showed no NF-κB nor p-IRF-3 nuclear translocation but rather MAPK and NLRP3-dependent inflammasome activation. The computational studies related a 3D shape of FP20 series with agonist binding properties inside the MD-2 pocket. FP20 displayed a CMC value lower than 5 µM in water, and small unilamellar vesicle (SUV) formation was observed in the biological activity concentration range. FP20 showed no toxicity in mouse vaccination experiments with OVA antigen and induced IgG production, thus indicating a promising adjuvant activity.
Asunto(s)
Adyuvantes de Vacunas , Receptor Toll-Like 4 , Ratones , Animales , Receptor Toll-Like 4/metabolismo , Adyuvantes Inmunológicos/farmacología , FN-kappa B/metabolismo , Vacunación , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismoRESUMEN
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
We recently reported on the activity of cationic amphiphiles in inhibiting TLR4 activation and subsequent production of inflammatory cytokines in cells and in animal models. Starting from the assumption that opportunely designed cationic amphiphiles can behave as CD14/MD-2 ligands and therefore modulate the TLR4 signaling, we present here a panel of amphiphilic guanidinocalixarenes whose structure was computationally optimized to dock into MD-2 and CD14 binding sites. Some of these calixarenes were active in inhibiting, in a dose-dependent way, the LPS-stimulated TLR4 activation and TLR4-dependent cytokine production in human and mouse cells. Moreover, guanidinocalixarenes also inhibited TLR4 signaling when TLR4 was activated by a non-LPS stimulus, the plant lectin PHA. While the activity of guanidinocalixarenes in inhibiting LPS toxic action has previously been related to their capacity to bind LPS, we suggest a direct antagonist effect of calixarenes on TLR4/MD-2 dimerization, pointing at the calixarene moiety as a potential scaffold for the development of new TLR4-directed therapeutics.
Asunto(s)
Calixarenos/química , Calixarenos/farmacología , Lectinas/farmacología , Lipopolisacáridos/farmacología , Receptor Toll-Like 4/metabolismo , Animales , Evaluación Preclínica de Medicamentos/métodos , Guanidina/química , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ligandos , Receptores de Lipopolisacáridos/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/antagonistas & inhibidoresRESUMEN
Adrenomedullin (AM) is a peptidic hormone that was isolated in 1993, the function of which is related to several diseases such as diabetes, hypertension, and cancer. Compound 1 is one of the first nonpeptidic small-molecule negative modulators of AM, identified in a high-throughput screen carried out at the National Cancer Institute. Herein we report the synthesis of a series of analogues of 1. The ability of the synthesized compounds to disrupt the binding between AM and its monoclonal antibody has been measured, together with surface plasmon resonance (SPR)-based binding assays as implemented with Biacore technology. These data were used to derive a three-dimensional quantitative structure-activity relationship (3D-QSAR) model, with a q(2) (LOO) value of 0.8240. This study has allowed us to identify relevant features for effective binding to AM: the presence of a hydrogen-bond donor group and an aromatic ring. Evaluation of the ability of selected compounds to modify cAMP production in Rat2 cells showed that the presence of a free carboxylic acid is essential for negative AM modulation.