Toll-like receptor 4 (TLR4) represents a central mediator of innate and adaptive immune responses in mammals. TLR4 activation in response to bacterial lipopolysaccharides (LPS) results in the rapid triggering of pro-inflammatory processes essential for optimal host immune responses. TLR4 activation mediated by LPS is a complex event which involves several proteins (lipid binding protein (LBP), cluster of differentiation 14 (CD14), and myeloid differentiation 2 (MD-2)) and it ends with the formation of the activated (TLR4/MD-2/LPS)2 complex. The TLR4 co-receptor MD-2 plays an important role in the interaction with LPS and subsequent TLR4 dimerization. MD-2 alone binds to LPS, whereas TLR4 alone does not. MD-2 is the ligand-binding component of the TLR4/MD-2 receptor complex. LPS binding to TLR4/MD-2 induces TLR4 dimerization; whereas TLR4 antagonists binding to TLR4/MD-2 does not induce TLR4 dimerization. Deregulated TLR4 activation is related to an impressively broad spectrum of disorders still lacking specific pharmacological treatment. These include autoimmune disorders, chronic inflammations, allergies, asthma, infectious and central nervous system diseases, cancer, and sepsis. The TLR4 inhibition by small molecules of synthetic and natural origin provides access to new TLR-based therapeutics targeting this large array of diseases. This thesis is part of an original structure-activity relationship (SAR) study on synthetic monosaccharide glycolipids in the context of TLR4 modulation. Thesis work focuses on the in vitro binding characterization of new synthetic monosaccharide glycolipids with the purified receptor MD-2. Pure and functional human MD-2 (hMD-2) protein for binding studies has been obtained by expression in yeast cells. Two different expression systems for the production of recombinant hMD-2 were tested: mammalian (HEK293T) and yeast cells (Pichia pastoris). Recovery of hMD-2 from the medium of yeast cells was optimized, achieving a concentration of recombinant hMD-2 of 30 μM. An ELISA was developed in order to compare the biological activity of the hMD-2 expressed in different hosts. hMD-2 from mammalian cells obtained the highest biological activity, followed by the hMD-2 expressed by P. pastoris. hMD-2 expressed by E. coli presented the lowest biological activity of the three. Due to the higher yield of recovery achieved, hMD-2 expressed in P. pastoris was used in four different types of binding experiments to assess its affinity for natural and synthetic molecules. The binding tests comprise two plate based ELISA with immobilized hMD-2, a fluorescence displacement assay and surface plasmon resonance (SPR) measurements. The two ELISA tests were based on: i) dose-dependent displacement of a monoclonal antibody from immobilized hMD-2. The antibody binds to hMD-2 in a region proximal to ligand binding site; ii) displacement of biotin-LPS from immobilized hMD-2. The fluorescence experiment was based on the displacement of the bis-ANS from hMD-2, whereas the SPR technique was used to study the direct interactions between small ligands and immobilized hMD-2. The obtained binding affinities for hMD-2 of the tested molecules (which turned out to be in the low μM range) mirror their biological activity in modulating TLR4 signaling and cytokine production in vitro in cell models. The results obtained from these in vitro cell-free studies indicate that the tested molecules bind to the hMD-2 pocket, with differences in the affinity values. These data allow a systematic study on SAR for TLR4 modulators, opening the way for the development of a new generation of drug hits and leads targeting directly TLR4 signaling.

Il toll-like receptor 4 (TLR4) rappresenta un mediatore centrale dell’immunità innata ed adattativa in mammiferi. L’attivazione di TLR4 in risposta al lipopolisaccaride (LPS) batterico induce un rapido innesco di processi pro-infiammatori essenziali per una risposta immunitaria ottimale. L’attivazione di TLR4 mediata da LPS è un meccanismo che coinvolge la partecipazione di diverse proteine e culmina con la formazione del complesso attivato (TLR4/MD-2/LPS)2. MD-2 è il co-rettore di TLR4, e svolge un importante ruolo nell’interazione con LPS e la susseguente dimerizzazione del TLR4. MD-2 è la componente che interagisce con il ligando (LPS) nel complesso recettoriale TLR4/MD-2. Il legame di LPS al complesso TLR4/MD-2 induce la dimerizzazione del TLR4; mentre gli antagonisti del TLR4 sono in grado di legare il complesso TLR4/MD-2 ma non inducono la dimerizzazione del TLR4. L’attivazione non regolata del TLR4 è correlata ad un’ampia serie di problematiche prive di un trattamento farmacologico specifico. Esse includono disordini autoimmuni, infiammazione cronica, allergie, asma, infezioni e malattie del sistema nervoso centrale, cancro, e setticemia. L’inibizione del TLR4 tramite l’uso di piccole molecole sintetiche o naturali può quindi rappresentare una via per lo sviluppo di nuove terapie contro questa vasta gamma di problematiche. Questa tesi è parte di un studio originale di relazione struttura-attività (SAR) svolto su glicolipidi monosaccaridici sintetici nel contesto della modulazione del TLR4. In particolare, essa si focalizza sulla caratterizzazione del legame in vitro di nuovi glicolipidi monosaccaridici sintetici con il recettore MD-2 purificato. Per gli studi di interazione la proteina MD-2 umana (hMD-2) purificata e funzionale è stata espressa in cellule di lievito. Due diversi sistemi di espressione per la produzione di hMD-2 ricombinante sono stati testati: mammifero (HEK293T) e cellule di lievito (Pichia pastoris). La purificazione di hMD-2 da lievito è stata ottimizzata ottenendo una concentrazione finale di hMD-2 purificato di 30 μM. Per confrontare l’attività biologica di hMD-2 espresso nei diversi microorganismi è stato sviluppato un ELISA. hMD-2 da cellule di mammifero ha ottenuto l’attività biologica più elevata, seguito da hMD-2 espresso in P. pastoris. hMD-2 da E. coli ha ottenuto l’attività biologica più bassa dei tre. Date le rese più elevate di purificazione in lievito, hMD-2 espresso in P. Pastoris è stato utilizzato nei quattro diversi tipi di esperimenti di legame per studiare l’affinità di molecole naturali e sintetiche. I test di legame comprendono due ELISA con hMD-2 immobilizzato, un saggio fluorescente di spiazzamento, e misure di risonanza plasmonica di superficie (SPR). I due test ELISA sono basati su: i) spiazzamento dose-dipendente di un anticorpo da hMD-2 immobilizzato. L’anticorpo lega hMD-2 in una regione in prossimità del sito di legame del ligando; ii) spiazzamento di LPS biotinilato da hMD-2 immobilizzato. L’esperimento di florescenza è basato sullo spiazzamento di bis-ANS da hMD-2. Mentre la tecnica SPR è stata utilizzata per studiare la diretta interazione tra le molecole e hMD-2 immobilizzato. L’affinità per hMD-2 delle molecole analizzate (che risulta essere nell’intervallo del basso μM) è in linea con i risultati di attività biologica e la produzione di citochine in vitro in modelli cellulari. I risultati ottenuti da questi studi in vitro sul recettore hMD-2 purificato evidenziano l’interazione delle molecole con la tasca idrofobica di hMD-2, presentando differenze nei valori di affinità. Questi dati generati permettono uno studio sistemico dei modulatori del TLR4, creando buone prospettive per lo sviluppo di una nuova generazione di farmaci hits e leads che agiscano direttamente sul recettore TLR4.

(2018). Production of recombinant human MD-2 and development of protein-ligand binding assays for the characterization of new TLR4 modulators. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).

Production of recombinant human MD-2 and development of protein-ligand binding assays for the characterization of new TLR4 modulators

ZAFFARONI, LENNY
2018

Abstract

Toll-like receptor 4 (TLR4) represents a central mediator of innate and adaptive immune responses in mammals. TLR4 activation in response to bacterial lipopolysaccharides (LPS) results in the rapid triggering of pro-inflammatory processes essential for optimal host immune responses. TLR4 activation mediated by LPS is a complex event which involves several proteins (lipid binding protein (LBP), cluster of differentiation 14 (CD14), and myeloid differentiation 2 (MD-2)) and it ends with the formation of the activated (TLR4/MD-2/LPS)2 complex. The TLR4 co-receptor MD-2 plays an important role in the interaction with LPS and subsequent TLR4 dimerization. MD-2 alone binds to LPS, whereas TLR4 alone does not. MD-2 is the ligand-binding component of the TLR4/MD-2 receptor complex. LPS binding to TLR4/MD-2 induces TLR4 dimerization; whereas TLR4 antagonists binding to TLR4/MD-2 does not induce TLR4 dimerization. Deregulated TLR4 activation is related to an impressively broad spectrum of disorders still lacking specific pharmacological treatment. These include autoimmune disorders, chronic inflammations, allergies, asthma, infectious and central nervous system diseases, cancer, and sepsis. The TLR4 inhibition by small molecules of synthetic and natural origin provides access to new TLR-based therapeutics targeting this large array of diseases. This thesis is part of an original structure-activity relationship (SAR) study on synthetic monosaccharide glycolipids in the context of TLR4 modulation. Thesis work focuses on the in vitro binding characterization of new synthetic monosaccharide glycolipids with the purified receptor MD-2. Pure and functional human MD-2 (hMD-2) protein for binding studies has been obtained by expression in yeast cells. Two different expression systems for the production of recombinant hMD-2 were tested: mammalian (HEK293T) and yeast cells (Pichia pastoris). Recovery of hMD-2 from the medium of yeast cells was optimized, achieving a concentration of recombinant hMD-2 of 30 μM. An ELISA was developed in order to compare the biological activity of the hMD-2 expressed in different hosts. hMD-2 from mammalian cells obtained the highest biological activity, followed by the hMD-2 expressed by P. pastoris. hMD-2 expressed by E. coli presented the lowest biological activity of the three. Due to the higher yield of recovery achieved, hMD-2 expressed in P. pastoris was used in four different types of binding experiments to assess its affinity for natural and synthetic molecules. The binding tests comprise two plate based ELISA with immobilized hMD-2, a fluorescence displacement assay and surface plasmon resonance (SPR) measurements. The two ELISA tests were based on: i) dose-dependent displacement of a monoclonal antibody from immobilized hMD-2. The antibody binds to hMD-2 in a region proximal to ligand binding site; ii) displacement of biotin-LPS from immobilized hMD-2. The fluorescence experiment was based on the displacement of the bis-ANS from hMD-2, whereas the SPR technique was used to study the direct interactions between small ligands and immobilized hMD-2. The obtained binding affinities for hMD-2 of the tested molecules (which turned out to be in the low μM range) mirror their biological activity in modulating TLR4 signaling and cytokine production in vitro in cell models. The results obtained from these in vitro cell-free studies indicate that the tested molecules bind to the hMD-2 pocket, with differences in the affinity values. These data allow a systematic study on SAR for TLR4 modulators, opening the way for the development of a new generation of drug hits and leads targeting directly TLR4 signaling.
PERI, FRANCESCO
FUSI, PAOLA ALESSANDRA
TLR4; MD-2; LPS; Immunità; Medicinal chemistry
TLR4; MD-2; LPS; Immunity; Medicinal chemistry
BIO/10 - BIOCHIMICA
English
9-lug-2018
BIOLOGIA E BIOTECNOLOGIE - 93R
30
2016/2017
open
(2018). Production of recombinant human MD-2 and development of protein-ligand binding assays for the characterization of new TLR4 modulators. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/207343
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