Systematic Evaluation of Regiochemistry and Lipidation of Aryl Trehalose Mincle Agonists.

The Macrophage-Inducible C-type Lectin receptor (Mincle) plays a critical role in innate immune recognition and pathology, and therefore represents a promising target for vaccine adjuvants. Innovative trehalose-based Mincle agonists with improved pharmacology and potency may prove useful in the development of Th17-mediated adaptive immune responses. Herein, we report on in vitro and in silico investigations of specific Mincle ligand-receptor interactions required for the effective receptor engagement and activation of Th17-polarizing cytokines. Specifically, we employed a library of trehalose benzoate scaffolds, varying the degree of aryl lipidation and regiochemistry that produce inflammatory cytokines in a Mincle-dependent fashion. In vitro interleukin-6 (IL-6) cytokine production by human peripheral blood mononuclear cells (hPBMCs) indicated that the lipid regiochemistry is key to potency and maximum cytokine output, with the tri-substituted compounds inducing higher levels of IL-6 in hPBMCs than the di-substituted derivatives. Additionally, IL-6 production trended higher after stimulation with compounds that contained lipids ranging from five to eight carbons long, compared to shorter (below five) or longer (above eight) carbon chains, across all the substitution patterns. An analysis of the additional cytokines produced by hPBMCs revealed that compound 4d, tri-substituted and five carbons long, induced significantly greater levels of interleukin-1ß (IL-1ß), tumor necrosis factor- α (TNF-α), interleukin-23 (IL-23), and interferon- γ (IFN-γ) than the other compounds tested in this study. An in silico assessment of 4d highlighted the capability of this analogue to bind to the human Mincle carbohydrate recognition domain (CRD) efficiently. Together, these data highlight important structure-activity findings regarding Mincle-specific cytokine induction, generating a lead adjuvant candidate for future formulations and immunological evaluations.

Promotion of Th17 Polarized Immunity via Co-Delivery of Mincle Agonist and Tuberculosis Antigen Using Silica Nanoparticles.

Adjuvants and immunomodulators that effectively drive a Th17-skewed immune response are not part of the standard vaccine toolkit. Vaccine adjuvants and delivery technologies that can induce Th17 or Th1/17 immunity and protection against bacterial pathogens, such as tuberculosis (TB), are urgently needed. Th17-polarized immune response can be induced using agonists that bind and activate C-type lectin receptors (CLRs) such as macrophage inducible C-type lectin (Mincle). A simple but effective strategy was developed for codelivering Mincle agonists with the recombinant Mycobacterium tuberculosis fusion antigen, M72, using tunable silica nanoparticles (SNP). Anionic bare SNP, hydrophobic phenyl-functionalized SNP (P-SNP), and cationic amine-functionalized SNP (A-SNP) of different sizes were coated with three synthetic Mincle agonists, UM-1024, UM-1052, and UM-1098, and evaluated for adjuvant activity in vitro and in vivo. The antigen and adjuvant were coadsorbed onto SNP via electrostatic and hydrophobic interactions, facilitating multivalent display and delivery to antigen presenting cells. The cationic A-SNP showed the highest coloading efficiency for the antigen and adjuvant. In addition, the UM-1098-adsorbed A-SNP formulation demonstrated slow-release kinetics in vitro, excellent stability over 12 months of storage, and strong IL-6 induction from human peripheral blood mononuclear cells. Co-adsorption of UM-1098 and M72 on A-SNP significantly improved antigen-specific humoral and Th17-polarized immune responses in vivo in BALB/c mice relative to the controls. Taken together, A-SNP is a promising platform for codelivery and proper presentation of adjuvants and antigens and provides the basis for their further development as a vaccine delivery platform for immunization against TB or other diseases for which Th17 immunity contributes to protection.

Vaccination with Mincle agonist UM-1098 and mycobacterial antigens induces protective Th1 and Th17 responses.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the top infectious killers in the world. The only licensed vaccine against TB, Bacille Calmette-Guérin (BCG), provides variable protection against pulmonary TB, especially in adults. Hence, novel TB vaccine approaches are urgently needed. Both Th1 and Th17 responses are necessary for protection against TB, yet effective adjuvants and vaccine delivery systems for inducing robust Th1 and Th17 immunity are lacking. Herein we describe a synthetic Mincle agonist, UM-1098, and a silica nanoparticle delivery system that drives Th1/Th17 responses to Mtb antigens. Stimulation of human peripheral blood mononuclear cells (hPBMCs) with UM-1098 induced high levels of Th17 polarizing cytokines IL-6, IL-1ß, IL-23 as well as IL-12p70, IL-4 and TNF-α in vitro. PBMCs from both C57BL/6 and BALB/c mice responded with a similar cytokine pattern in vitro and in vivo. Importantly, intramuscular (I.M.) vaccination with UM-1098-adjuvanted TB antigen M72 resulted in significantly higher antigen-specific IFN-γ and IL-17A levels in C57BL/6 wt mice than Mincle KO mice. Vaccination of C57BL/6 wt mice with immunodominant Mtb antigens ESAT6/Ag85B or M72 resulted in predominantly Th1 and Th17 responses and induced antigen-specific serum antibodies. Notably, in a virulent Mtb challenge model, vaccination with UM-1098 adjuvanted ESAT6/Ag85B or M72 significantly reduced lung bacterial burden when compared with unvaccinated mice and protection occurred in the absence of pulmonary inflammation. These data demonstrate that the synthetic Mincle agonist UM-1098 induces strong Th1 and Th17 immunity after vaccination with Mtb antigens and provides protection against Mtb infection in mice.

Diamino Allose Phosphates: Novel, Potent, and Highly Stable Toll-like Receptor 4 Agonists.

Most known synthetic toll-like receptor 4 (TLR4) agonists are carbohydrate-based lipid-A mimetics containing several fatty acyl chains, including a labile 3-O-acyl chain linked to the C-3 position of the non-reducing sugar known to undergo cleavage impacting stability and resulting in loss of activity. To overcome this inherent instability, we rationally designed a new class of chemically more stable synthetic TLR4 ligands that elicit robust innate and adaptive immune responses. This new class utilized a diamino allose phosphate (DAP) scaffold containing a nonhydrolyzable 3-amide bond instead of the classical 3-ester. Accordingly, the DAPs have significantly improved thermostability in aqueous formulations and potency relative to other known natural and synthetic TLR4 ligands. Furthermore, the DAP analogues function as potent vaccine adjuvants to enhance influenza-specific antibodies in mice and provide protection against lethal influenza virus challenges. This novel set of TLR4 ligands show promise as next-generation vaccine adjuvants and stand-alone immunomodulators.

Synthesis and Biological Evaluation of Trehalose-based Bi-aryl Derivatives as C-type Lectin Ligands.

The identification of Mincle as the C-type lectin receptor on innate immune cells responsible for binding TDM and the realization that this receptor could be key to productive vaccines for mycobacterial infection has raised interest in the development of synthetic Mincle ligands as novel adjuvants. We recently reported on the synthesis and evaluation of Brartemicin analog UM-1024 that demonstrated Mincle agonist activity, exhibiting potent Th1/Th17 adjuvant activity that was greater than that of trehalose dibehenate (TDB). Our pursuit to understand Mincle/ligand relationships and improve the pharmacologic properties of the ligands has expanded and continues to reveal new and exciting structure activity relationships. Herein we report the synthesis of novel bi-aryl trehalose derivatives in good to excellent yields. These compounds were evaluated for their ability to engage the human Mincle receptor and tested for the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure-activity relationship (SAR) of these novel bi-aryl derivatives revealed that bi-aryl trehalose ligand 3D showed relatively high potency in cytokine production in comparison to trehalose glycolipid adjuvant TDB and the natural ligand TDM and induced dose-dependent, Mincle selective stimulation in hMincle HEK reporter cells. Also, through computational studies, we provide an insight into the potential mode of binding of 6,6'-Biaryl trehalose compounds on human Mincle receptor.

Design of Trehalose-Based Amide/Sulfonamide C-type Lectin Receptor Signaling Compounds.

Mincle agonists have been shown to induce inflammatory cytokine production, such as tumor necrosis factor-alpha (TNF) and promote the development of a Th1/Th17 immune response that might be crucial to development of effective vaccination against pathogens such as Mycobacterium tuberculosis. As an expansion of our previous work, a library of 6,6'-amide and sulfonamide α,α-d-trehalose compounds with various substituents on the aromatic ring was synthesized efficiently in good to excellent yields. These compounds were evaluated for their ability to activate the human C-type lectin receptor Mincle by the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure-activity relationship (SAR) of these novel trehalose diamides and sulfonamides revealed that aryl amide-linked trehalose compounds demonstrated improved activity and relatively high potency cytokine production compared to the Mincle ligand trehalose dibehenate adjuvant (TDB) and the natural ligand trehalose dimycolate (TDM) inducing dose-dependent and human-Mincle-specific stimulation in a HEK reporter cell line.

Co-adsorption of synthetic Mincle agonists and antigen to silica nanoparticles for enhanced vaccine activity: A formulation approach to co-delivery.

To date there is no clinically approved adjuvant to drive a protective T-helper cell 17 (Th17) immune response against Mycobacterium tuberculosis (Mtb). Trehalose Dimycolate (TDM) is a glycolipid molecule found in the cell wall of Mtb and similar species. Our team has discovered novel synthetic TDM derivatives that target Mincle receptors and when presented on the surface of amine functionalized silica nanoparticles (A-SNPs) adopt the requisite supramolecular structure for Mincle receptor agonism. Here we describe the preparation and characterization methods for these critical silica nanoparticles (SNPs) co-loaded with Mincle agonists (MAs) and a model antigen. In this work, A-SNPs with a particle diameter of 246 ± 11 nm were prepared and examined for co-adsorption of two synthetic MAs along with ovalbumin (OVA). Due to the insolubility of the studied MAs in aqueous environment, aggregation of the MAs made separation of the adjuvant-loaded A-SNPs from the free-form MAs via centrifugation very challenging. To facilitate separation, we synthesized modified SNPs with comparable amine surface functionalization to the original A-SNPs, but with a superparamagnetic iron oxide core (M-A-SNPs), to allow for magnetic separation. We also substituted Alexa Fluor 488-labeled ovalbumin (AF 488 OVA) for the un-tagged OVA to improve the sensitivity of our quantitation method. A RP-HPLC method was developed to simultaneously determine the amount of adsorption of both the Mincle adjuvant and the model antigen to the A-SNPs. AF488 OVA demonstrated higher than 90% adsorption, with or without the co-adsorption of MAs. Likewise, MAs exhibited higher than 80% adsorption in the presence or absence of antigen. The developed formulations were tested in vitro using murine RAW cells and human peripheral blood mononuclear cells, exhibiting good cytokine induction in both cell lines. Results from these studies indicate that A-SNPs could be used as a customizable presentation platform to co-deliver antigens along with different MAs of varying structural features and biophysical properties.

6,6'-Aryl trehalose analogs as potential Mincle ligands.

6,6'-Aryl trehalose derivatives have been synthesized with a view towards identifying novel Th-17-inducing vaccine adjuvants based on the high affinity Mincle ligand Brartemicin. The initial structure-activity relationships of these novel trehalose-based compounds were investigated. All compounds have been evaluated for their ability to engage the Mincle receptor and induce a potential pro-Th17 cytokine profile from human peripheral blood mononuclear cells based on IL-6 production in human peripheral blood mononuclear cells. The preliminary biological characterization of the designed analogs presented in this paper should aid in the future design and testing of more affine ligands that may foster the discovery of novel adjuvants with improved pharmacological properties.

Aryl Trehalose Derivatives as Vaccine Adjuvants for Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) continues to be a major health threat worldwide, and the development of Mtb vaccines could play a pivotal role in the prevention and control of this devastating epidemic. Th17-mediated immunity has been implicated in disease protection correlates of immune protection against Mtb. Currently, there are no approved adjuvants capable of driving a Th17 response in a vaccine setting. Recent clinical trial results using trehalose dibehenate have demonstrated a formulation-dependant proof of concept adjuvant system CAF01 capable of inducing long-lived protection. We have discovered a new class of Th17-inducing vaccine adjuvants based on the natural product Brartemicin. We synthesized and evaluated the capacity of a library of aryl trehalose derivatives to drive immunostimulatory reresponses and evaluated the structure-activity relationships in terms of the ability to engage the Mincle receptor and induce production of innate cytokines from human and murine cells. We elaborated on the structure-activity relationship of the new scaffold and demonstrated the ability of the lead entity to induce a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells and demonstrated efficacy in generating antibodies in combination with tuberculosis antigen M72 in a mouse model.

Species-Specific Structural Requirements of Alpha-Branched Trehalose Diester Mincle Agonists.

Despite the ever present need for an effective Mycobacterium tuberculosis (Mtb) vaccine, efforts for development have been largely unsuccessful. Correlates of immune protection against Mtb are not wholly defined, but Th1 and likely Th17 adaptive immune responses have been demonstrated to be necessary for vaccine-mediated protection. Unfortunately, no approved adjuvants are able to drive a Th17 response, though recent clinical trials with CAF01 have demonstrated proof of concept. Herein we present the discovery and characterization of a new class of potential Th17-inducing vaccine adjuvants, alpha-branched trehalose diester molecules (αTDE). Based off the Mtb immunostimulatory component trehalose dimycolate (TDM), we synthesized and evaluated the immunostimulatory capacity of a library of structural derivatives. We evaluated the structure activity relationship of the compounds in relation to chain length and engagement of the Mincle receptor, production of innate cytokines from human and murine cells, and a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells. Murine cells displayed more structural tolerance, engaging and responding to a wide array of compound chain lengths. Interestingly, human cells displayed a unique specificity for ester chains between 5 and 14 carbons for maximal immune stimulating activity. Evaluation of two distinct αTDEs, B16 and B42, in concert with a recombinant Mtb antigen demonstrated their ability to augment a Th17 immune response against a Mtb antigen in vivo. Collectively this data describes the species-specific structural requirements for maximal human activity of alpha-branched trehalose diester compounds and demonstrates their capacity to serve as potent Th17-inducing adjuvants.

Immunoregulatory Activity of the Natural Product Laminarin Varies Widely as a Result of Its Physical Properties.

Ligation of Dectin-1 by fungal glucans elicits a Th17 response that is necessary for clearing many fungal pathogens. Laminarin is a (1→3, 1→6)-ß-glucan that is widely reported to be a Dectin-1 antagonist, however, there are reports that laminarin is also a Dectin-1 agonist. To address this controversy, we assessed the physical properties, structure, purity, Dectin-1 binding, and biological activity of five different laminarin preparations from three different commercial sources. The proton nuclear magnetic resonance analysis indicated that all of the preparations contained laminarin although their molecular mass varied considerably (4400-34,400 Da). Two of the laminarins contained substantial quantities of very low m.w. compounds, some of which were not laminarin. These low m.w. moieties could be significantly reduced by extensive dialysis. All of the laminarin preparations were bound by recombinant human Dectin-1 and mouse Dectin-1, but the affinity varied considerably, and binding affinity did not correlate with Dectin-1 agonism, antagonism, or potency. In both human and mouse cells, two laminarins were Dectin-1 antagonists and two were Dectin-1 agonists. The remaining laminarin was a Dectin-1 antagonist, but when the low m.w. moieties were removed, it became an agonist. We were able to identify a laminarin that is a Dectin-1 agonist and a laminarin that is Dectin-1 antagonist, both of which are relatively pure preparations. These laminarins may be useful in elucidating the structure and activity relationships of glucan/Dectin-1 interactions. Our data demonstrate that laminarin can be either a Dectin-1 antagonist or agonist, depending on the physicochemical properties, purity, and structure of the laminarin preparation employed.

Characterization of TRIF selectivity in the AGP class of lipid A mimetics: role of secondary lipid chains.

TLR4 agonists that favor TRIF-dependent signaling and the induction of type 1 interferons may have potential as vaccine adjuvants with reduced toxicity. CRX-547 (4), a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of lipid A mimetics possessing three (R)-3-decanoyloxytetradecanoyl groups and d-relative configuration in the aglycon, selectively reduces MyD88-dependent signaling resulting in TRIF-selective signaling, whereas the corresponding secondary ether lipid 6a containing (R)-3-decyloxytetradecanoyl groups does not. In order to determine which secondary acyl groups are important for the reduction in MyD88-dependent signaling activity of 4, the six possible ester/ether hybrid derivatives of 4 and 6a were synthesized and evaluated for their ability to induce NF-κB in a HEK293 cell reporter assay. An (R)-3-decanoyloxytetradecanoyl group on the 3-position of the d-glucosamine unit was found to be indispensable for maintaining low NF-κB activity irrespective of the substitutions (decyl or decanoyl) on the other two secondary positions. These results suggest that the carbonyl group of the 3-secondary lipid chain may impede homodimerization and/or conformational changes in the TLR4-MD2 complex necessary for MyD88 binding and pro-inflammatory cytokine induction.

The 'Ethereal' nature of TLR4 agonism and antagonism in the AGP class of lipid A mimetics.

To overcome the chemical and metabolic instability of the secondary fatty acyl residues in the AGP class of lipid A mimetics, the secondary ether lipid analogs of the potent TLR4 agonist CRX-527 (2) and TLR4 antagonist CRX-526 (3) were synthesized and evaluated along with their ester counterparts for agonist/antagonist activity in both in vitro and in vivo models. Like CRX-527, the secondary ether lipid 4 showed potent agonist activity in both murine and human models. Ether lipid 5, on the other hand, showed potent TLR4 antagonist activity similar to CRX-526 in human cell assays, but did not display any antagonist activity in murine models and, in fact, was weakly agonistic. Glycolipids 2, 4, and 5 were synthesized via a new highly convergent method utilizing a common advanced intermediate strategy. A new method for preparing (R)-3-alkyloxytetradecanoic acids, a key component of ether lipids 4 and 5, is also described.

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 4: functionalization of the benzopyran A-ring.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER receptor subtypes alpha and beta in opposite orientations. We have used structure based drug design to show that this unique phenomena can be exploited via substitution at the 8-position of the benzopyran A-ring to disrupt binding to ERalpha, thus improving ERbeta subtype selectivity. X-ray cocrystal structures with ERalpha and ERbeta are supportive of this approach to improve selectivity in this structural class.

Preparation of 8-Amido-2-dimethylamino-1,2,3,4-tetrahydro-2-dibenzofurans and several fluorinated derivatives via [3,3]-sigmatropic rearrangement of O-aryloximes.

Methodology to prepare 8-amido-2-amino-1,2,3,4-tetrahydro-2-dibenzofurans, analogues with a fluorine substituent incorporated in the 6-, 7-, and 9-positions, and a difluorinated analogue with fluorines in the 6- and 9-positions is described. The tetrahydrodibenzofuran ring systems are prepared by acid-catalyzed [3,3]-sigmatropic rearrangement of O-aryloximes. Regioselective reactions to prepare the requisite O-aryloxime intermediates from commercially available fluorobenzene derivatives are discussed.