Modulators of Toll-like Receptors-4 and -2

Abstract

The toxicity of Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) resides in its structurally highly conserved glycolipid component called lipid A. The major goal of the first project was to further explore structure-activity relationships in small-molecules that would sequester LPS by binding to the lipid A moiety, so that it may find application in the prophylaxis or adjunctive therapy of Gram-negative sepsis. Several guanylhydrazones had earlier been identified in rapid-throughput screens as potent LPS binders. It was desirous to examine if grafting the guanylhydrazone functionality on the scaffold of a lead N-alkyl polyamine compound would afford greater LPS sequestration potency. In the first project, a congeneric set of guanylhydrazone analogues were synthesized and evaluated for LPS-sequestering potency. It was found that a C16 alkyl substitution was optimal in the N-alkylguanylhydrazone series; a homospermine analogue with the terminal amine N-alkylated with a C16 chain with the other terminus of the molecule bearing an unsubstituted guanylhydrazone moiety was marginally more active suggesting very slight, if any, steric effects. Neither C16 analogue was significantly more active than the N-C16-alkyl or N-C16-acyl compounds that we had characterized earlier, indicating that basicity of the phosphate-recognizing cationic group is not a determinant of LPS sequestration activity. 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. 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. In the second project, I 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 were maximally active in reporter gene assays using human TLR2. The Cys-Ser dipeptide unit represents the minimal part-structure, but the stereochemistry of neither amino residues of the dipeptide was found to be a critical determinant of activity. The thioether bridge between the diacyl and dipeptide units was determined to be crucial, and replacement by an ether bridge resulted in a dramatic decrease in activity. Moreover, the replacement of the two ester-liked C16 hydrocarbons by ether or amide linkages led to a dramatic loss in activity, while the replacement of one of the ester-linked fatty acyl moiety (internal) with an amide linkage remained partially active.