Department of Medicinal Chemistry
Sunil A. David
Innate Immunity and Toll-like Receptors (TLRs):
Toll-like receptors (TLRs) are pattern recognition receptors present on diverse cell types that recognize specific molecular patterns present in molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). There are 10 TLRs in the human genome; these are trans-membrane proteins with an extracellular domain having leucine-rich repeats (LRR) and a cytosolic domain called the Toll/IL-1 receptor (TIR) domain. The ligands for these receptors are 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 (recognized by TLR9), and profilin present on uropathogenic bacteria (TLR 11). The activation of TLRs by their cognate ligands leads to production of inflammatory cytokines, and up-regulation of MHC molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer (NK) cells, in addition to priming and amplifying T-, and B-cell effector functions (adaptive immune responses). Thus, TLR stimuli serve to link innate and adaptive immunity and can therefore be exploited as powerful adjuvants in eliciting both primary and anamnestic immune responses.
The N-terminus 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. We have examined in the 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. 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 ether bridge results in a dramatic decrease in activity.
Nod1-Agonistic Gamma-Glutamyl-diaminopimelic Acid Derivatives:
Members of the NLR (nucleotide-binding and oligomerization domain, leucine rich repeats) family of intracellular pathogen associated receptors play an important role in recognizing pathogens, and trigger NF-kappa B-mediated proinflammatory cytokine release. An SAR of C12-gamma-D-Glu-DAP was conducted. Transcriptomal profiling using whole human blood showed a dominant upregulation of Class II helical cytokines of the interleukin (IL)-10 superfamily, including IL-19, IL-20, IL-22, and IL-24, which may explain the pronounced Th2-polarizing activity of these compounds. Further analyses of gene transcripts implicate cell signaling mediated by Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1). These results may explain the hitherto unknown mechanism of synergy between Nod1- and TLR-agonists, and are likely to be of value in designing vaccine adjuvants.
Structure-Activity Relationships in Human Toll-like Receptor 7-Active Imidazoquinolines:
TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. A systematic exploration of N1-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 N1 and C2 substituents led to the identification of an extremely active TLR7-agonistic compound with an EC50 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. 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.
A TLR7-active N1-(4-aminomethyl)benzyl substituted imidazoquinoline 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. Covalent conjugation of the isothiocyanate derivative to alpha-lactalbumin could be achieved under mild, non-denaturing conditions, in a controlled manner and with full preservation of antigenicity. The self-adjuvanting alpha-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.
TLR7- and -8 Antagonism in Imidazoquinoline dimers:
Chronic immune activation is a hallmark of progressive HIV infection. Recent reports point to the engagement of toll-like receptor 7 (TLR7) and -9 by viral RNA as contributing to the activation of innate immune responses, which drive viral replication leading to immune exhaustion. The only known class of TLR7 antagonists is single-stranded phosphorothioate oligonucleotides, which has been demonstrated to inhibit immune activation in human and Rhesus macaque in in vitro models. The availability of a selective and potent small-molecule TLR7 antagonist should allow the evaluation of potential benefits of suppression of TLR7-mediated immune activation in HIV/AIDS.We synthesized and evaluated TLR7 and TLR8 modulatory activities of dimeric constructs of imidazoquinolines linked at the C2, C4, C8, and N1-aryl positions. Dimers linked at the C4, C8 and N1-aryl positions were agonistic at TLR7; only the N1-aryl dimer with a 12-carbon linker was found to be dual TLR7/8 agonistic. The imidazoquinoline dimers linked at C2 position showed antagonistic activities at TLR7 and TLR8; the C2 dimer with a propylene spacer was found to be maximally antagonistic at both TLR7 and TLR8, and its activities were preserved in secondary screens employing human blood.
Development of small-molecule endotoxin sequestering agents.
Sepsis, otherwise referred to as "blood poisoning" is a serious clinical problem, the incidence of which continues to rise in the US and worldwide despite advances in antimicrobial chemotherapy. The primary trigger in Gram-negative sepsis is endotoxin, a lipopolysaccharide (LPS) constituent of the outer membrane of all Gram-negative bacteria. The structurally highly conserved glycolipid called lipid A is the active moiety of LPS. Lipid A is composed of a hydrophilic, bis-phosphorylated di-glucosamine backbone, and a hydrophobic polyacyl domain. The bis-anionic, amphiphilic nature of lipid A enables it to interact with a variety of cationic hydrophobic ligands, including polymyxin B, a toxic peptide antibiotic which binds to lipid A and neutralizes endotoxicity. Having determined the structural basis of the interaction of polymyxin B with lipid A, our long-term goal has been to rationally design non-peptidic, nontoxic, small-molecule LPS-sequestrants. Our efforts began with defining the central pharmacophore that determined LPS-recognition and -neutralization properties in small molecules, which led to the discovery of a novel lipopolyamine lead, DS-96. DS-96 is an effective LPS-neutralizer, rivaling polymyxin B in a panel of vitro assays, as well as in protecting animals against endotoxicosis. Structure-activity relationships are being explored in an effort to rationally design endotoxin sequestering agents.