Rajalakshmi Balakrishna

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.

Structure-Activity Relationships in Human Toll-like Receptor 7-Active Imidazoquinoline Analogues

Engagement of toll-like receptors serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. A structure-activity study was conducted on the TLR7-agonistic imidazoquinolines, starting with 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol as a lead. Modifications of the secondary amine of the C2 ethylaminomethylene side chain are poorly tolerated. The 4-amino group must be retained for activity. Replacement of the imidazole ring of the scaffold with triazole or cyclic urea led to complete loss of activity. A systematic exploration of N(1)-benzyl-C2-alkyl substituents showed a very distinct relationship between alkyl length and TLR7-agonistic potency with the optimal compound bearing a C2-n-butyl group. Transposition of the N(1) and C2 substituents led to the identification of an extremely active TLR7-agonistic compound with an EC(50) value of 8.6 nM. The relative potencies in human TLR7-based primary reporter gene assays were paralleled by interferon-alpha induction activities in whole human blood models.

Toward self-adjuvanting subunit vaccines: model peptide and protein antigens incorporating covalently bound toll-like receptor-7 agonistic imidazoquinolines.

Toll-like receptor (TLR)-7 agonists show prominent Th1-biased immunostimulatory activities. A TLR7-active N(1)-(4-aminomethyl)benzyl substituted imidazoquinoline 1 served as a convenient precursor for the syntheses of isothiocyanate and maleimide derivatives for covalent attachment to free amine and thiol groups of peptides and proteins. 1 was also amenable to direct reductive amination with maltoheptaose without significant loss of activity. Covalent conjugation of the isothiocyanate derivative 2 to α-lactalbumin could be achieved under mild, non-denaturing conditions, in a controlled manner and with full preservation of antigenicity. The self-adjuvanting α-lactalbumin construct induced robust, high-affinity immunoglobulin titers in murine models. The premise of covalently decorating protein antigens with adjuvants offers the possibility of drastically reducing systemic exposure of the adjuvant, and yet eliciting strong, Th1-biased immune responses.

Antibacterial activities of Groebke-Blackburn-Bienaymé-derived imidazo[1,2-a]pyridin-3-amines.

We sought to explore the imidazo[1,2-a]pyridin-3-amines for TLR7 (or 8)-modulatory activities. This chemotype, readily accessed via the Groebke-Blackburn-Bienaymé multi-component reaction, resulted in compounds that were TLR7/8-inactive, but exhibited bacteriostatic activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). To investigate the mechanism of antibacterial activity of this new chemotype, a resistant strain of S. aureus was generated by serially passaging the organism in escalating doses of the most active analogue. A comparison of minimum inhibitory concentrations (MICs) of known bacteriostatic agents in wild-type and resistant strains indicates a novel mechanism of action. Structure-activity relationship studies have led to the identification of positions on the scaffold for additional structural modifications that should allow for the introduction of probes designed to examine cognate binding partners and molecular targets, while not significantly compromising antibacterial potency.

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 Adjuvanticity of a Pure TLR7-Agonistic Imidazoquinoline Dendrimer

Engagement of toll-like receptors (TLRs) serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. We synthesized a dendrimeric molecule bearing six units of a potent TLR7/TLR8 dual-agonistic imidazoquinoline to explore if multimerization of TLR7/8 would result in altered activity profiles. A complete loss of TLR8-stimulatory activity with selective retention of the TLR7-agonistic activity was observed in the dendrimer. This was reflected by a complete absence of TLR8-driven proinflammatory cytokine and interferon (IFN)-γ induction in human PBMCs, with preservation of TLR7-driven IFN-α induction. The dendrimer was found to be superior to the imidazoquinoline monomer in inducing high titers of high-affinity antibodies to bovine α-lactalbumin. Additionally, epitope mapping experiments showed that the dendrimer induced immunoreactivity to more contiguous peptide epitopes along the amino acid sequence of the model antigen.

Structure–Activity Relationships in Human Toll-like Receptor 2-Specific 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. We had previously determined that at least one acyl group of optimal length (C(16)) and an appropriately orientated ester carbonyl group is essential for TLR2-agonistic activity. We now show that these structurally simpler analogues display agonistic activities with human, but not murine, TLR2. SAR studies on the monoacyl derivatives show that the optimal acyl chain length is C(16), and aryl substituents are not tolerated. A variety of alkyl and acyl substituents on the cysteine amine were examined. All N-alkyl derivatives were inactive. In contradistinction, short-chain N-acyl analogues were found to be highly active, with a clear dependence on the chain length. A cysteine N-acetyl analogue was found to be the most potent (EC(50): 1 nM), followed by the N-butyryl analogue. The N-acetyl analogue is human TLR2-specific, with its potency comparable to that of PAM(2)CS.

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.

Structural Correlates of Antibacterial and Membrane-Permeabilizing Activities in Acylpolyamines

ABSTRACT A homologous series of mono- and bis-acyl polyamines with varying acyl chain lengths originally synthesized for the purpose of sequestering lipopolysaccharide were evaluated for antimicrobial activity to test the hypothesis that these bis-cationic amphipathic compounds may also bind to and permeabilize intact gram-negative bacterial membranes. Some compounds were found to possess significant antimicrobial activity, mediated via permeabilization of bacterial membranes. Structure-activity relationship studies revealed a strong dependence of the acyl chain length on antimicrobial potency and permeabilization activity. Homologated spermine, bis-acylated with C8 or C9 chains, was found to profoundly sensitize Escherichia coli to hydrophobic antibiotics such as rifampin. Nonspecific cytotoxicity is a potential drawback of these membranophilic compounds. However, the surface activity of these cationic amphipaths is strongly attenuated under physiological conditions via binding to serum albumin. Significant antibacterial activity is still retained in the presence of physiological concentrations of human serum albumin, suggesting that these compounds may serve as leads in the development of novel adjuncts to conventional antimicrobial chemotherapy.