Toll-like receptor (TLR)-7 and -8 modulatory activities of dimeric imidazoquinolines.

Toll-like receptors (TLRs) are pattern recognition receptors that recognize specific molecular patterns present in molecules that are broadly shared by pathogens but are structurally distinct from host molecules. The TLR7-agonistic imidazoquinolines are of interest as vaccine adjuvants given their ability to induce pronounced Th1-skewed humoral responses. Minor modifications on the imidazoquinoline scaffold result in TLR7-antagonistic compounds which may be of value in addressing innate immune activation-driven immune exhaustion observed in HIV. We describe the syntheses and evaluation of TLR7 and TLR8 modulatory activities of dimeric constructs of imidazoquinoline linked at the C2, C4, C8, and N(1)-aryl positions. Dimers linked at the C4, C8, and N(1)-aryl positions were agonistic at TLR7; only the N(1)-aryl dimer with a 12-carbon linker was dual TLR7/8 agonistic. Dimers linked at C2 position showed antagonistic activities at TLR7 and TLR8; the C2 dimer with a propylene spacer was maximally antagonistic at both TLR7 and TLR8.

Potent adjuvantic activity of a CCR1-agonistic bis-quinoline.

A bis-quinoline compound, (7-chloro-N-(4-(7-chloroquinolin-4-ylamino)butyl)quinolin-4-amine; RE-660) was found to have C-C chemokine receptor type 1 (CCR1)-agonistic properties. RE-660 displayed strong adjuvantic activity in mice when co-administered with bovine α-lactalbumin used as a model subunit protein antigen. RE-660 evoked a balanced Th1 (IgG2)/Th2 (IgG1) antibody profile, and the quality of antibodies elicited by the bis-quinoline was found to be superior to that evoked by glucopyranosyl lipid A by surface plasmon resonance experiments. No evidence of proinflammatory activity was observed in human blood ex vivo models. In preliminary acute toxicity studies, the compound was found to be of lower toxicity than chloroquine in mice, and was non-mutagenic in an Ames screen.

Structure-activity relationships in toll-like receptor 2-agonists leading to simplified monoacyl lipopeptides.

Toll-like receptor 2-agonistic lipopeptides typified by S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM(2)CS) compounds are potential vaccine adjuvants. In continuation of previously reported structure-activity relationships on this chemotype, we have determined that at least one acyl group of optimal length (C(16)) and an appropriately oriented ester carbonyl group is essential for TLR2-agonistic activity. The spacing between one of the palmitoyl ester carbonyl and the thioether is crucial to allow for an important H-bond, which observed in the crystal structure of the lipopeptide:TLR2 complex; consequently, activity is lost in homologated compounds. Penicillamine-derived analogues are also inactive, likely due to unfavorable steric interactions with the carbonyl of Ser 12 in TLR2. The thioether in this chemotype can be replaced with a selenoether. Importantly, the thioglycerol motif can be dispensed with altogether and can be replaced with a thioethanol bridge. These results have led to a structurally simpler, synthetically more accessible, and water-soluble analogue possessing strong TLR2-agonistic activities in human blood.

Structure-activity relationships in nucleotide oligomerization domain 1 (Nod1) agonistic γ-glutamyldiaminopimelic acid derivatives.

N-acyl-γ-glutamyldiaminopimelic acid is a prototype ligand for Nod1. We report a detailed SAR of C(12)-γ-D-Glu-DAP. Analogues with glutaric or γ-aminobutyric acid replacing the glutamic acid show greatly attenuated Nod1-agonistic activity. Substitution of the meso-diaminopimelic (DAP) acid component with monoaminopimelic acid, L- or D-lysine, or cadaverine also results in reduced activity. The free amine on DAP is crucial. However, the N-acyl group on the D-glutamyl residue can be substituted with N-alkyl groups with full preservation of activity. The free carboxylates on the DAP and Glu components can also be esterified, resulting in more lipophilic but active analogues. Transcriptomal profiling showed a dominant up-regulation of IL-19, IL-20, IL-22, and IL-24, which may explain the pronounced Th2-polarizing activity of these compounds and also implicate cell signaling mediated by TREM-1. These results may explain the hitherto unknown mechanism of synergy between Nod1 and TLR agonists and are likely to be useful in designing vaccine adjuvants.

Syntheses of fluorescent imidazoquinoline conjugates as probes of Toll-like receptor 7.

Toll-like receptor (TLR)-7 agonists show prominent immunostimulatory activities. The synthesis of a TLR7-active N(1)-(4-aminomethyl)benzyl substituted imidazoquinoline 5d served as a convenient precursor for the covalent attachment of fluorophores without significant loss of activity. Fluorescence microscopy experiments show that the fluorescent analogues are internalized and distributed in the endosomal compartment. Flow cytometry experiments using whole human blood show differential partitioning into B, T, and natural killer (NK) lymphocytic subsets, which correlate with the degree of activation in these subsets. These fluorescently-labeled imidazoquinolines will likely be useful in examining the trafficking of TLR7 in immunological synapses.

Immunoprofiling toll-like receptor ligands: Comparison of immunostimulatory and proinflammatory profiles in ex vivo human blood models.

There is a pressing need for the development of novel, safe and effective adjuvants. The recent discovery and characterization of pathogen-associated molecular pattern (PAMP)-recognizing elements such as the Toll-like, NOD-like and RIG-like receptors, has brought into sharp focus the role of PAMPs in bridging the innate and adaptive immune responses, and a detailed understanding of the immunostimulatory vis-à-vis proinflammatory activities could lead to the development of effective adjuvants, monophosphoryl lipid A being an excellent example. We describe in this paper a series of hierarchical assays that were employed to characterize TLR agonists in vitro including primary TLR-reporter assays, secondary indices of immune activation, and tertiary screens characterizing transcriptomal activation patterns to identify optimal immunostimulatory chemotypes. The evaluation of representative members of known human TLR agonists demonstrate that TLR2, -4, -5 and -7 agonists were immunostimulatory. TLR7 agonists were extremely immunostimulatory, stimulating nearly all subsets of lymphocytes without inducing proinflammatory cytokine responses. The TLR5 agonist, flagellin, while immunostimulatory, was also highly proinflammatory. These results suggest that TLR agonists other than lipid A-like chemotypes could be developed into potential adjuvants, and that this series of hierarchical assays could be adapted to rapidly identify in large libraries, compounds with adjuvantic potential that lack proinflammatory responses.

Structure-activity relationships in toll-like receptor-2 agonistic diacylthioglycerol lipopeptides.

The N-termini of bacterial lipoproteins are acylated with a (S)-(2,3-bisacyloxypropyl)cysteinyl residue. Lipopeptides derived from lipoproteins activate innate immune responses by engaging Toll-like receptor 2 (TLR2) and are highly immunostimulatory and yet without apparent toxicity in animal models. The lipopeptides may therefore be useful as potential immunotherapeutic agents. Previous structure-activity relationships in such lipopeptides have largely been obtained using murine cells, and it is now clear that significant species-specific differences exist between human and murine TLR responses. We have examined in detail the role of the highly conserved Cys residue as well as the geometry and stereochemistry of the Cys-Ser dipeptide unit. (R)-Diacylthioglycerol analogues are maximally active in reporter gene assays using human TLR2. The Cys-Ser dipeptide unit represents the minimal part-structure, but its stereochemistry was found not to be a critical determinant of activity. The thioether bridge between the diacyl and dipeptide units is crucial, and replacement by an oxoether bridge results in a dramatic decrease in activity.

Synthesis of a highly water-soluble derivative of amphotericin B with attenuated proinflammatory activity.

Amphotericin B (AmB), a well-known polyene antifungal agent, displays a marked tendency to self-associate and, as a consequence, exhibits very poor solubility in water. The therapeutic index of AmB is low and is associated with significant dose-related nephrotoxicity, as well as acute, infusion-related febrile reactions. Reports in the literature indicate that the toxicity of AmB may be related to the physical state of the drug. Reaction of AmB in dimethylformamide with bis(dimethylaminopropyl)carbodiimide yielded an unexpected N-alkylguanidine/N-acylurea bis-adduct of AmB which was highly water-soluble. The absorption spectrum of the AmB derivative in water indicated excellent monomerization, and the antifungal activities of reference AmB and its water-soluble derivative against Candida albicans were found to be virtually identical. Furthermore, the water-soluble adduct is significantly less active in engaging TLR4, which would suggest that the adduct may be less proinflammatory.

Structure-activity relationships of lipopolysaccharide sequestration in N-alkylpolyamines.

We have previously shown that simple N-acyl or N-alkyl polyamines bind to and sequester Gram-negative bacterial lipopolysaccharide, affording protection against lethality in animal models of endotoxicosis. Several iterative design-and-test cycles of SAR studies, including high-throughput screens, had converged on compounds with polyamine scaffolds which have been investigated extensively with reference to the number, position, and length of acyl or alkyl appendages. However, the polyamine backbone itself had not been explored sufficiently, and it was not known if incremental variations on the polymethylene spacing would affect LPS-binding and neutralization properties. We have now systematically explored the relationship between variously elongated spermidine [NH(2)-(CH(2))(3)-NH-(CH(2))(4)-NH(2)] and norspermidine [NH(2)-(CH(2))(3)-NH-(CH(2))(3)-NH(2)] backbones, with the N-alkyl group being held constant at C(16) in order to examine if changing the spacing between the inner secondary amines may yield additional SAR information. We find that the norspermine-type compounds consistently showed higher activity compared to corresponding spermine homologues.

Potential adjuvantic properties of innate immune stimuli.

Toll-like receptors (TLRs) are a family of conserved pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns and serve as primary sensors of the innate immune system. Ten members of the TLR family have so far been identified in the human genome. The ligands for these receptors are structurally highly conserved microbial molecules such as lipopolysaccharides (LPS) (recognized by TLR4), lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5), single stranded RNA (TLR7 and TLR8), double-stranded RNA (TLR3), CpG motif-containing DNA (TLR9) and profilin present on uropathogenic bacteria (TLR 11). Complementing the TLRs are the nucleotide-binding domain (NOD), leucine rich repeat containing family (or Nod-like Receptors, NLRs), which detect muramylpeptides released from bacterial peptidoglycan (PGN) in the intracytoplasmic compartment, as well as the retinoic-acid-inducible protein 1 (RIG-I-like receptors; RLRs) which sense single-stranded RNA of viral origin. The activation of PRRs by their cognate ligands leads to production of inflammatory cytokines, upregulation of MHC molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer cells, in addition to priming and amplifying antigen-specific T-, and B-cell effector functions. Thus, these stimuli serve to link innate and adaptive immunity and can therefore be exploited as powerful adjuvants in eliciting both primary and anamnestic immune responses. This review summarizes what is currently known about the immunopotentiatory and adjuvantic activities of innate immune stimuli.

Structure-activity relationships of antimicrobial and lipoteichoic acid-sequestering properties in polyamine sulfonamides.

We have recently confirmed that lipoteichoic acid (LTA), a major constituent of the gram-positive bacterial surface, is the endotoxin of gram-positive bacteria that induces proinflammatory molecules in a Toll-like receptor 2 (TLR2)-dependent manner. LTA is an anionic amphipath whose physicochemical properties are similar to those of lipopolysaccharide (LPS), which is found on the outer leaflet of the outer membranes of gram-negative organisms. Hypothesizing that compounds that sequester LPS could also bind to and inhibit LTA-induced cellular activation, we screened congeneric series of polyamine sulfonamides which we had previously shown effectively neutralized LPS both in vitro and in animal models of endotoxemia. We observed that these compounds do bind to and neutralize LTA, as reflected by the inhibition of TLR2-mediated NF-kappaB induction in reporter gene assays. Structure-activity studies showed a clear dependence of the acyl chain length on activity against LTA in compounds with spermine and homospermine scaffolds. We then sought to examine possible correlations between the neutralizing potency toward LTA and antimicrobial activity in Staphylococcus aureus. A linear relationship between LTA sequestration activity and antimicrobial activity for compounds with a spermine backbone was observed, while all compounds with a homospermine backbone were equally active against S. aureus, regardless of their neutralizing potency toward LTA. These results suggest that the number of protonatable charges is a key determinant of the activity toward the membranes of gram-positive bacteria. The development of resistance to membrane-active antibiotics has been relatively slower than that to conventional antibiotics, and it is possible that compounds such as the acylpolyamines may be useful clinically, provided that they have an acceptable safety profile and margin of safety. A more detailed understanding of the mechanisms of interactions of these compounds with LPS and LTA, as well as the gram-negative and -positive bacterial cell surfaces, will be instructive and should allow the rational design of analogues which combine antisepsis and antibacterial properties.

Structure-activity relationships of lipopolysaccharide sequestration in guanylhydrazone-bearing lipopolyamines.

The toxicity of gram-negative bacterial endotoxin (lipopolysaccharide, LPS) resides in its structurally highly conserved glycolipid component called lipid A. Our major goal has been to develop small-molecules that would sequester LPS by binding to the lipid A moiety, so that it could be useful for the prophylaxis or adjunctive therapy of gram-negative sepsis. We had previously identified in rapid-throughput screens several guanylhydrazones as potent LPS binders. We were desirous of examining if the presence of the guanylhydrazone (rather than an amine) functionality would afford greater LPS sequestration potency. In evaluating a congeneric set of guanylhydrazone analogues, we find that C(16) alkyl substitution is optimal in the N-alkylguanylhydrazone series; a homospermine analogue with the terminal amine N-alkylated with a C(16) chain with the other terminus of the molecule bearing an unsubstituted guanylhydrazone moiety is marginally more active, suggesting very slight, if any, steric effects. Neither C(16) analogue is significantly more active than the N-C(16)-alkyl or N-C(16)-acyl compounds that we had characterized earlier, indicating that basicity of the phosphate-recognizing cationic group, is not a determinant of LPS sequestration activity.

Comparison of the immunostimulatory and proinflammatory activities of candidate Gram-positive endotoxins, lipoteichoic acid, peptidoglycan, and lipopeptides, in murine and human cells.

The role of lipopolysaccharide (LPS) in the pathogenesis of Gram-negative septic shock is well established. The corresponding proinflammatory and immunostimulatory molecule(s) on the Gram-positive bacteria is less well understood, and its identification and characterization would be a key prerequisite in designing specific sequestrants of the Gram-positive endotoxin(s). We report in this paper the comparison of NF-kappaB-, cytokine- and chemokine-inducing activities of the TLR2 ligands, lipoteichoic acid (LTA), peptidoglycan (PGN), and lipopeptides, to LPS, a prototype TLR4 agonist, in murine macrophage cell-lines as well as in human blood. In murine cells, di- and triacyl liopopeptides are equipotent in their NF-kappaB inducing activity relative to LPS, but elicit much lower proinflammatory cytokines. However, both LPS and the lipopeptides potently induce the secretion of a pattern of chemokines that is suggestive of the engagement of a TLR4-independent TRIF pathway. In human blood, although the lipopeptides induce p38 MAP kinase phosphorylation and CD11b upregulation in granulocytes at ng/ml concentrations, they do not elicit proinflammatory cytokine production even at very high doses; LTA, however, activates neutrophils and induces cytokine secretion, although its potency is considerably lower than that of LPS, presumably due to its binding to plasma proteins. We conclude that, in human blood, the pattern of immunostimulation and proinflammatory mediator production elicited by LTA parallels that of LPS.

Interactions of a designed peptide with lipopolysaccharide: Bound conformation and anti-endotoxic activity.

Designed peptides that would selectively interact with lipopolysaccharide (LPS) or endotoxin and fold into specific conformations could serve as important scaffolds toward the development of antisepsis compounds. Here, we describe solution structure of a designed amphipathic peptide, H(2)N-YVKLWRMIKFIR-CONH(2) (YW12D) in complex with endotoxin as determined by transferred nuclear Overhauser effect spectroscopy. The conformation of the isolated peptide is highly flexible, but undergoes a dramatic structural stabilization in the presence of LPS. Structure calculations reveal that the peptide presents two amphipathic surfaces in its bound state to LPS whereby each surface is characterized by two positive charges and a number of aromatic and/or aliphatic residues. ITC data suggests that peptide interacts with two molecules of lipid A. In activity assays, YW12D exhibits neutralization of LPS toxicity with very little hemolysis of red blood cells. Structural and functional properties of YW12D would be applicable in designing low molecular weight non-toxic antisepsis molecules.

Controlling plasma protein binding: structural correlates of interactions of hydrophobic polyamine endotoxin sequestrants with human serum albumin.

Hydrophobically substituted polyamine compounds, particularly N-acyl or N-alkyl derivatives of homospermine, are potent endotoxin (lipopolysaccharide) sequestrants. Despite their polycationic nature, the aqueous solubilites are limited owing to the considerable overall hydrophobicity contributed by the long-chain aliphatic substituent, but solubilization is readily achieved in the presence of human serum albumin (HSA). We desired first to delineate the structural basis of lipopolyamine-albumin interactions and, second, to explore possible structure-activity correlates in a well-defined, congeneric series of N-alkyl and -acyl homospermine lead compounds. Fluorescence spectroscopic and isothermal titration calorimetry (ITC) results indicate that these compounds appear to bind to HSA via occupancy of the fatty-acid binding sites on the protein. The acyl and carbamate compounds bind HSA the strongest; the ureido and N-alkyl analogues are significantly weaker, and the branched alkyl compound is weaker still. ITC-derived dissociation constants are weighted almost in their entirety by enthalpic deltaH terms, which is suggestive that the polarizability of the carbonyl groups facilitate, at least in large part, their interactions with HSA. The relative affinities of these lipopolyamines toward HSA is reflected in discernible differences in apparent potencies of LPS-sequestering activity under experimental conditions requiring physiological concentrations of HSA, and also of in vivo pharmacodynamic behavior. These results are likely to be useful in designing analogues with varying pharmacokinetic profiles.