Design, synthesis and bioconjugation of TLR4 ligands to understand and modulate innate immunity processes

Toll-like receptors (TLRs) are among the first receptors activated during host-pathogen interactions. They act as key mediators in the pathogen-associated molecular patterns (PAMPs) detection and are responsible for the innate and adaptive immune responses in mammal. Belonging to this family, Toll-like receptor 4 (TLR4) is the mammalian sensor of bacterial endotoxin, lipopolysaccharide (LPS). Its activation results in the rapid triggering of pro-inflammatory processes essential for optimal host immune responses. TLR4 activation is a complex event which involves several proteins (LBP, CD14, and MD-2) and ends with the dimerization as an activated (TLR4/MD-2/LPS)2 complex. Dysregulated TLR4 activation is related to an impressively broad spectrum of disorders still lacking specific pharmacological treatment, including autoimmune disorders, chronic inflammations, allergies, asthma, infectious and central nervous system diseases, cancer, and sepsis. TLR4 modulation by small molecules of synthetic and natural origin provides access to new TLR-based therapeutics. Indeed, compounds able to block TLR4 activation (antagonists) are promising drug candidates against thee pathologies while TLR4 activating compounds (agonists) may be use as vaccines adjuvants and antitumoral agents. The work of this PhD thesis is described in four papers. The aim of the thesis is to study the processes and requirements leading to TLR4 activation/inhibition. The first study, Chap. IX, focuses on the contribution of carbohydrates to bacterial endotoxin (lipopolysaccharide, LPS, lipooligosaccharide, LOS, and lipid A) activity and in the switch from TLR4 agonism to antagonism. In particular, the structure-activity relationship and contribution of core saccharides 3-deoxy-D-manno-octulosonic acid (Kdo) and heptosyl-2-keto-3-deoxy-octulosonate (Hep) to TLR4/MD-2 binding and activation by LPS and LOS have been investigated in detail. This study allows the rational design of new structures of potential TLR4 modulators. These compounds were projected by computer-assisted design and their binding to MD-2 was evaluated through docking studies. A set of the most promising compounds was defined. They are composed of a glucosamine-bis-succinyl core (two carboxylate groups as phosphates bioisosteres) linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to MD-2 was studied by four independent methods using functional recombinant human MD-2 protein purified. It was demonstrated that all compounds bound to MD-2 with similar affinities and inhibited, in a concentration-dependent manner, the LPS-stimulated TLR4 signaling and cytokine production in human and murine cells.