Activation of Toll-like receptor 4 (TLR4) and subsequent intracellular signaling in response to minute amounts of circulating endotoxins (Gram-negative bacterial lipopolysaccharides, LPS), results in the rapid triggering of proinflammatory processes necessary for optimal host immune responses to invading Gram-negative bacteria in mammalians. TLR4 does not bind directly to LPS, and TLR4 activation by endotoxin is a complex event, involving the participation of other LPS-binding proteins — namely LBP, CD14, and MD-2 — and ending with the formation of the activated (TLR4•MD-2•LPS)2 complex. In particular, CD14 was the first identified Pattern Recognition Receptor (PRR) that binds directly to LPS, and chaperones the formation of the (TLR4•MD-2•LPS)2 complex. CD14 is also required for endotoxin-induced TLR4 endocytosis and relocalization of the entire LPS receptor complex into the endosome, where a second signaling pathway initiates. Excessively potent and deregulated TLR4 activation and signaling causes severe syndromes such as septic shock, associated with a high mortality (50–70%), and organ-specific damages. Efficient and selective TLR4 antagonists with chemical structures simpler than that of lipid A are therefore required for the development of potential new drugs with a wide array of medical and pharmacological applications (from sepsis to CNS pathologies). In this thesis, the synthesis of three monosaccharide LPS mimetics based on a glucosamine core linked to two fatty acid chains and bearing one or two phosphate groups is described. Two of them, each with one phosphate group, resulted quite active in inhibiting LPS-induced TLR4 signaling and cytokine production in HEK-Blue™ cells and murine macrophages. The compound with two phosphate groups, however, was found to be more active in efficiently inhibiting TLR4 signal in both cell types. The direct interaction between this compound and the MD-2 receptor was investigated by NMR spectroscopy and molecular modeling/docking analysis. This compound also interacts directly with the CD14 receptor, stimulating its internalization by endocytosis. The dual targeting of MD-2 and CD14, accompanied by good solubility in water and lack of toxicity, suggests the use of monosaccharide 3 as a lead compound for the development of drugs directed against TLR4-related syndromes. A derivatization of this compound was also started in order to couple it to a fluorescent moiety or to a Gadolinium-chelating agent, for microscopy and MRI studies respectively.

L’attivazione del Toll-Like Receptor 4 (TLR4) e il conseguente signaling intracellulare in risposta a esigue quantità di endotossine (lipopolisaccaridi di batteri Gram-negativi, LPS) presenti nel circolo sanguigno porta al rapido innesco di processi pro-infiammatori necessari per una risposta immunitaria ottimale dell’organismo ospite contro l’invasione di batteri Gram-negativi. Il TLR4 non lega però direttamente i LPS e la sua attivazione da parte dell’endotossina è un processo complesso che prevede la partecipazione di altre proteine che legano i LPS (LBP, CD14 e MD-2) e porta infine alla formazione del complesso attivato (TLR4•MD-2•LPS)2. In particolare, CD14 è stato il primo recettore che lega LPS ad essere scoperto e sembra che favorisca la formazione del complesso (TLR4•MD-2•LPS)2. CD14 è anche necessario per l’endocitosi di TLR4 indotta da LPS e la localizzazione dell’intero complesso recettoriale per l’endotossina negli endosomi, da dove parte un secondo pathway del segnale. Un’attivazione di TLR4 eccessivamente potente e sregolata causa però gravi sindromi come lo shock settico, associato a un’alta mortalità (50-70%) e a danni agli organi anche molto gravi. Sono perciò necessari degli antagonisti (inibitori) di TLR4 efficaci e selettivi con una struttura chimica più semplice di quella del LPS, per lo sviluppo di potenziali nuovi farmaci con un’ampia gamma di applicazioni mediche e farmacologiche (dalla sepsi alle patologie del sistema nervoso). In questa tesi è descritta la sintesi di monosaccaridi mimetici del LPS basati su un core di glucosammina legata a due catene di acidi grassi che portano uno o due gruppi fosfato. Di questi, i due composti con un solo fosfato hanno mostrato una certa attività come inibitori dell’attivazione di TLR4 e della produzione di citochine in cellule HEK-Blue® e in macrofagi murini. Il composto con due gruppi fosfato è risultato invece molto più efficace nell’inibire il segnale di TLR4 in entrambi i modelli cellulari. L’interazione diretta tra questo composto e il recettore MD-2 è stata investigata mediante spettroscopia NMR e analisi bioinformatiche (molecular modelling/docking). Questo composto interagisce direttamente anche con il recettore CD14, inducendone l’internalizzazione per endocitosi. Il targeting sia di CD14 che di MD-2, unito a una buona solubilità in acqua e all’assenza di tossicità, rendono il monosaccaride difosfato presentato in questa tesi un possibile lead compound per lo sviluppo di farmaci contro le sindromi collegate al TLR4. E’ stata inoltre avviata una funzionalizzazione di questo composto in modo da coniugarlo a una molecola fluorescente o a un chelante del gadolinio per studi, rispettivamente, di microscopia o imaging mediante risonanza magnetica.

(2014). Design, synthesis and biological characterization of new small-molecule TLR4 modulators. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2014).

Design, synthesis and biological characterization of new small-molecule TLR4 modulators

CIGHETTI, ROBERTO
2014

Abstract

Activation of Toll-like receptor 4 (TLR4) and subsequent intracellular signaling in response to minute amounts of circulating endotoxins (Gram-negative bacterial lipopolysaccharides, LPS), results in the rapid triggering of proinflammatory processes necessary for optimal host immune responses to invading Gram-negative bacteria in mammalians. TLR4 does not bind directly to LPS, and TLR4 activation by endotoxin is a complex event, involving the participation of other LPS-binding proteins — namely LBP, CD14, and MD-2 — and ending with the formation of the activated (TLR4•MD-2•LPS)2 complex. In particular, CD14 was the first identified Pattern Recognition Receptor (PRR) that binds directly to LPS, and chaperones the formation of the (TLR4•MD-2•LPS)2 complex. CD14 is also required for endotoxin-induced TLR4 endocytosis and relocalization of the entire LPS receptor complex into the endosome, where a second signaling pathway initiates. Excessively potent and deregulated TLR4 activation and signaling causes severe syndromes such as septic shock, associated with a high mortality (50–70%), and organ-specific damages. Efficient and selective TLR4 antagonists with chemical structures simpler than that of lipid A are therefore required for the development of potential new drugs with a wide array of medical and pharmacological applications (from sepsis to CNS pathologies). In this thesis, the synthesis of three monosaccharide LPS mimetics based on a glucosamine core linked to two fatty acid chains and bearing one or two phosphate groups is described. Two of them, each with one phosphate group, resulted quite active in inhibiting LPS-induced TLR4 signaling and cytokine production in HEK-Blue™ cells and murine macrophages. The compound with two phosphate groups, however, was found to be more active in efficiently inhibiting TLR4 signal in both cell types. The direct interaction between this compound and the MD-2 receptor was investigated by NMR spectroscopy and molecular modeling/docking analysis. This compound also interacts directly with the CD14 receptor, stimulating its internalization by endocytosis. The dual targeting of MD-2 and CD14, accompanied by good solubility in water and lack of toxicity, suggests the use of monosaccharide 3 as a lead compound for the development of drugs directed against TLR4-related syndromes. A derivatization of this compound was also started in order to couple it to a fluorescent moiety or to a Gadolinium-chelating agent, for microscopy and MRI studies respectively.
PERI, FRANCESCO
Toll-Like Receptor 4; Lipopolysaccharide; LPS; CD14; MD-2; Sepsis; Septic Shock; Medicinal Chemistry; Organic Synthesis
CHIM/08 - CHIMICA FARMACEUTICA
English
6-feb-2014
Scuola di dottorato di Scienze
BIOTECNOLOGIE INDUSTRIALI - 15R
26
2012/2013
open
(2014). Design, synthesis and biological characterization of new small-molecule TLR4 modulators. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2014).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/50753
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