Cu-CPT22

CU-CPT22 can compete with the synthetic triacylated lipoprotein (Pam3CSK4) binding to TLR1/2 with high inhibitory activity and specificity. The inhibition constant (Ki) is 0.41 ± 0.07 µM. CU-CPT22 inhibits TLR1/2 signaling without affecting other TLRs, showing it is highly selective in intact cells. It has no significant cytotoxicity at various concentrations up to 100 µM in RAW 264.7 cells using MTT assay. Kinase profiling shows that CU-CPT22 demonstrates minimal non-specific inhibition against a panel of 10 representative kinases (PDGFRB, MET, DDR2, SRC, MAPK1, PAK1, AKT1, PKC-γ, CAMK1, and PLK4). CU-CPT22 suppresses TLR1/2-mediated inflammation response. It inhibits about 60% of TNF-α and 95% of IL-1β at 8 µM in the RAW 264.7 cells^[1].

MUTZ-LCs are seeded in alpha medium without supple-ments and exposed to different microbial and pro-inflammatorystimuli for 24 h (2.5 × 10⁵ cells/ml): 1 μg/ml Pam3CSK4, 1 μg/mlPam2CSK4, 1 μg/ml poly(A:U), 1 μg/ml poly(I:C), 1 μg/ml ultrapurelipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4, 50 ng/ml rh-TNF-α, 30 ng/ml rh-IL-1β, or a cytokine maturation cocktail(CMC) consisting of 50 ng/ml rh-TNF-α, 25 ng/ml rh-IL-1β,100 ng/ml rh-IL-6 and 1 μg/ml PGE2. Cytokines produced in E. coli, contained low endotoxinlevels (≤1.0 EU/μg cytokine) as determined by Limulus Amebo-cyte Lysate (LAL) assay. As control MUTZ-LCs (2.5 × 10⁵ cells/ml) are maintained for 24 hor 48 h in alpha medium only. The TLR2/1 antagonist CU-CPT22 is applied 1 h before stimulation with Pam3CSK4 and Pam2CSK4, respectively.

Cu-CPT22 administered before myocardial infarction (MI) significantly suppresses MI-induced upregulation of kidney injury molecule-1 (KIM-1), TLR2, TLR4, MyD88, and chemokine (C-C motif) ligand 2 levels and activation of NF-κB, whereas neutrophil gelatinase-associated lipocalin (NGAL) levels and IL-6 and TNF-α expression levels are unchanged^[2].