Pam3CSK4 is a synthetic triacylated lipopeptide (LP) which is similar to bacterial LPs that have an acylated amino terminus. Bacterial LPs are found in both Gram-positive and Gram-negative bacteria, and they are members of a family of proinflammatory cell wall components. Pam3CSK4 can efficiently activate the proinflammatory transcription factor NF-κB, a process that is mediated by Toll-like receptors 2 (TLR2) which combines with TLR1 via their cytoplasmic structural domain to drive the signaling amplification and lead to the activation of NF-κB. TLR1 and TLR2 can also recognize the characteristic structural triacylated fatty acids of the bacterial LPS.
Toll-like receptors (TLRs)
TLR2 is mainly distributed on the surface of antigen-presenting cells, such as dendritic cells. As a classical type I transmembrane protein, consisting of an extracellular region with leucine-rich repeat sequences (LRRs), a transmembrane region and an intracellular region with a Toll-IL-1 receptor (TIR) structural domain.
TLR2 recognizes lipopeptides, lipoproteins and other structures in microorganisms by forming heterodimers with TLR1 or TLR6, and then the intracellular TIR structural domain recruits the junctional protein myeloid differentiation factor 88 (Myd88), which activates the downstream signaling molecules IL-1-related protein kinases (IRAKs), tumor necrosis factor receptor associated factor 6 (TRAF6), to activate the nuclear transcription factor NF κB, induce the production of various cytokines, such as IL-6 and TNF-α. Therefore, activation of TLR2 can rapidly initiate innate immunity while regulating the specific immunity in vivo.
TLRs play an indispensable role in the immune defense as the pathway of the innate immune response and the regulator of specific immunity, particularly TLR2 has become an important target for immune regulation. Efficient activation of TLR2 is an effective means to enhance the immune response, therefore, TLR2 agonists have shown great potential for vaccine adjuvant, antitumor, antibacterial and immune-based disease treatment.
Pam3CSK4, as a kind of TLRs agonists, is a synthetic lipopeptide containing three palmitoyl groups derived from bacterial lipoproteins observed the function of activating lymphocytes. The reported cocrystal structure of complexes of Pam3CSK4 with human TLR2/hTLR1 showed that Pam3CSK4 binds to the heterodimer TLR2/TLR1, and two palmitoyl aliphatic chains containing ester bonds are bound to the hydrophobic pocket of TLR2, while the other palmitoyl aliphatic chain with an amide bond is bound to the hydrophobic pocket of TLR1. The four lysine residues play a role in maintaining the active conformation and in regulating the physicochemical properties, with two of the lysine amino groups involved in the formation of hydrogen bonds.
Application of Pam3CSK4
Pam3CSK4 has been shown to be an effective adjuvant for a variety of vaccines, applied in the sublingual allergy vaccine, flu vaccine and leishmaniasis vaccine. For example, sublingual immunotherapy (SLIT) has been established in humans as a safe and efficacious treatment for type I respiratory allergies. Testing in a model of SLIT in mice, Pam3CSK4 is the potent inducer of IL-12p35 and IL-10 gene expression in murine bone marrow-derived dendritic cells, as well as in purified oral myeloid dendritic cells. Pam3CSK4 is able to inducing IFN-γ and IL-10 secretion by T cells and is a valid adjuvant for sublingual allergy vaccines. In addition, Pam3CSK4 is proven greatly alleviate nasal allergy symptoms and decreased total inflammatory cells and eosinophils in bronchoalveolar lavage fluid (BALF) through experiments in rhinitis mice. It has also been shown that TLR2a promotes the production of the inflammatory cytokines TNF-α and IL-6 in dogs, which suggest the importance of Pam3CSK4 as a possible immunomodulator for Canine leishmaniosis.
Antitumor
Immune checkpoint inhibitors such as anti–CTLA-4 antibody are widely accepted therapeutic options for many cancers. TLRs ligand Pam3CSK4 has been reported that combining with anti–CTLA-4 antibody to treat mouse model of melanoma, which enhanced antitumor immune responses both qualitatively and quantitatively over anti–CTLA-4 alone. Pam3CSK4 increased Fcγ receptor IV expression on macrophages, leading to antibody-dependent macrophage-mediated depletion of regulatory T cells in the tumor microenvironment and increasing efficacy of anti–CTLA-4 antibody in the combination treatment.
Antibacterial
A study has shown that comparing with classical method of generating neutrophils directly from murine bone marrow, pretreatment with Pam3CSK4 enhanced the phagocytic and killing activities against methicillin-resistant Staphylococcus aureus (MRSA) by the granulocyte macrophage-colony stimulating factor (GM-CSF) induced neutrophils. Furthermore, Pam3CSK4 pretreatment enhanced iNOS, CRAMP, TNF-α, IL-1β, IL-10, and IL-6 expression.