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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.

TLR4 è il recettore dell’immunità innata che riconosce selettivamente LPS di batteri Gram-negativi che hanno superato le barriere fisiche ed anatomiche del nostro organismo. TLR4 risponde a LPS batterici e a diversi ligandi endogeni in associazione con la proteina MD-2 ed innesca così le risposte immunitarie e infiammatorie necessarie per combattere l’infezione. I recettori LBP e CD14 sono coinvolti nel processo di riconoscimento di LPS e nel trasferimento di LPS dagli aggregati in soluzione al complesso TLR4-MD-2. TLR4 è stato recentemente collegato a molte importanti patologie che mancano ancora di un trattamento farmacologico specifico: sepsi e shock settico, cancro, patologie da infiammazioni acute o croniche, SLA, arteriosclerosi, allergie, asma, disordini cardiovascolari, diabete. Composti in grado di bloccare l’attivazione del TLR4 (antagonisti) sono quindi dei potenziali candidati per la cura di queste patologie, mentre composti in grado di stimolare TLR4 (agonisti) possono essere usati come agenti antitumorali o come adiuvanti vaccinali. La struttura chimica del lipide A (la parte biologicamente attiva di LPS) è stata semplificata in glicolipidi sintetici basati sulla struttura del Lipide X, il precursore biosintetico del Lipide A. Diversi fra questi composti hanno dimostrato di possedere attività agonistica o antagonistica su TLR4. Degli 8 adiuvanti vaccinali attualmente approvati o in valutazione clinica, tre sono monosaccaridi derivati delle strutture parziali del Lipide A. Tuttavia, le relazioni struttura-attività ed il passaggio da antagonismo ad agonismo o viceversa per i modulatori di TLR4 monosaccaridici non sono state ancora chiarificati. Con l’obiettivo di ottenere nuovi modulatori monosaccaridi di TLR4 (agonisti o antagonisti), allo scopo di esplorare le SAR e con altresì il fine di studiare le proprietà di binding con il co-recettore di TLR4 MD-2, durante il lavoro di questa tesi di dottorato sono stati progettate e sintetizzate 9 innov, TLR4 is the innate immunity receptor which selectively recognizes LPS from the Gram-negative bacteria that eluded the physical and anatomical barriers of our organisms. TLR4 responds to bacterial LPS and to different endogenous ligands in association with the protein adaptor MD-2 and triggers the immune and inflammatory responses. The receptors LBP and CD14 are involved in the LPS recognition process and in the transfer of LPS from aggregates in solution to TLR4-MD-2 complex . TLR4 has recently been related to many important pathologies still lacking specific pharmacological treatment: from diseases caused by bacterial infection such as sepsis and septic shock, to cancer and acute or chronic inflammatory diseases, atherosclerosis, allergies, asthma, cardiovascular disorder, diabetes. Compounds able to block TLR4 activation (antagonists) are drug candidates against these pathologies, compounds active in stimulating TLR4 (agonists) may be used as antitumoral agents or vaccine adjuvants. The chemical structure of lipid A (the biologically active part of LPS) has been simplified into synthetic glycolipids resembling lipid X, a biosynthetic precursor of lipid A. These compounds retained part of the agonist or antagonist activity on TLR4. Of the 8 vaccine adjuvants currently approved or in clinical evaluation, 3 are monosaccharide derivatives of Lipid A partial structures. However, SAR and the switch from agonism to antagonism for monosaccharide TLR4 modulators have not been completely clarified yet, nor their activities have been clearly rationalized with respect to MD-2 binding. With the aim of obtaining new monosaccharide-based TLR4 modulators (antagonists or agonists), to explore their SAR and to study their MD-2 binding properties, during this PhD thesis 9 new monosaccharide molecular simplifications of Lipid A have been projected and synthesized: FP7, FP11, FP111, AM158, AM173, AMX1, AM246248-d1, AM246248-d2 and AM241. This series of molecules has been projected by

This study examines the effect of co-administration of antimicrobial peptides and the synthetic glycolipid FP7, which is active in inhibiting inflammatory cytokine production caused by TLR4 activation and signaling. The co-administration of two lipopolysaccharide (LPS)-neutralizing peptides (a cecropin A–melittin hybrid peptide and a human cathelicidin) enhances by an order of magnitude the potency of FP7 in blocking the TLR4 signal. Interestingly, this is not an additional effect of LPS neutralization by peptides, because it also occurs if cells are stimulated by the plant lectin phytohemagglutinin, a non-LPS TLR4 agonist. Our data suggest a dual mechanism of action for the peptides, not exclusively based on LPS binding and neutralization, but also on a direct effect on the LPS-binding proteins of the TLR4 receptor complex. NMR experiments in solution show that peptide addition changes the aggregation state of FP7, promoting the formation of larger micelles. These results suggest a relationship between the aggregation state of lipid A-like ligands and the type and intensity of the TLR4 response.

A thiophene-based donor-acceptor phenothiazine dye has been functionalized with a peripheral glucose unit (PTZ-GLU) to bust its affinity to water and enhance dye-sensitized photogeneration of hydrogen. Compared to the corresponding alkyl derivative (PTZ-ALK), as well as the common hydrophilic triethylene glycol substitution (PTZ-TEG), the sugar derivative shows a lower contact angle; PTZ-GLU performed twice more efficient than PTZ-TEG in the photogeneration of hydrogen in terms of evolved gas and turnover number.

A thiophene-based donor-acceptor phenothiazine dye has been functionalized with a peripheral glucose unit (PTZ-GLU) to bust its affinity to water and enhance dye-sensitized photogeneration of hydrogen. Compared to the corresponding alkyl derivative (PTZ-ALK), as well as the common hydrophilic triethylene glycol substitution (PTZ-TEG), the sugar derivative shows a lower contact angle; PTZ-GLU performed twice more efficient than PTZ-TEG in the photogeneration of hydrogen in terms of evolved gas and turnover number.