Stereoisomeric Pam<sub>2</sub>CS based TLR2 agonists: synthesis, structural modelling and activity as vaccine adjuvants.
Kaur A, Piplani S, Kaushik D, Fung J, Sakala IG, Honda-Okubo Y, Mehta SK, Petrovsky N, Salunke DB.
Lipopeptides including diacylated Pam<sub>2</sub>CSK<sub>4</sub> as well as triacylated Pam<sub>3</sub>CSK<sub>4</sub> act as ligands of toll-like receptor (TLR)-2, a promising target for the development of vaccine adjuvants. The highly investigated Pam<sub>2</sub>CSK<sub>4</sub> and Pam<sub>3</sub>CSK<sub>4</sub>, despite their aqueous solubility have not performed well as vaccine adjuvants which may be attributable to potential denaturation of protein antigens by these cationic surfactant-like lipopeptides. In the present investigation, we synthesized (<i>R</i>), (<i>S</i>) and racemic Pam<sub>2</sub>CS(OMe) analogs and their <i>N</i>-acetyl derivatives without the tetralysine component to systematically investigate the effect of stereochemistry at the thio-glycerol lipopeptide core of these lipopeptide based TLR2 agonists. The resulting compounds were compared using TLR2 reporter cell-based assays and the ability of the synthesized lipopeptides to stimulate cytokine production (IL-6, IL-10 and TNF-α) by freshly collected human PBMCs and CD40 and CD86 expressions by mouse spleen cells was also investigated. Notably, few synthesized lipopeptides were found to be potent TLR2/6 agonists, inducing cytokine production and upregulating CD40 and CD86 expressions. The TLR2/6 agonistic lipopeptides were further assessed for vaccine adjuvant effects in mice. The results confirmed that the <i>R</i>-stereochemistry at the thio-glycerol lipopeptide core was preferred for maximal TLR2/6 activity, as reflected in Th1 immune deviation, higher antibody levels and enhanced vaccine protection against a lethal influenza challenge.