Screening, Identification And Application Of Targeting TLR2Peptides

Toll-like receptor2plays a fundamental role in pathogen recognition and activation of innate immunity as a pattern recognition receptor. Recent studies found that TLR2activity participated with the pathogenesis of autoimmune diseases, tumor carcinogenesis and cardio-cerebrovascular diseases. In this study, enhanced expression of toll like receptor2(TLR2) was observed in acute myeloid leukemia (AML) cells. Therefore, TLR2and its signalling components are promising target candidates for drug development. Our study established a TLR2receptor-based cell screening model and new TLR2agonist and cell penetrating peptide were identified from the Ph.D.TM-7Phage Display Peptide Library. Chimeric peptides composed of the TLR2-binding motif linked to therapeutic effect sequences target acute myeloid leukemia or tumors. The major research results are shown as following:1. Establishment, identification and application of TLR2receptor-based cell screening model.To establish a TLR2receptor-based cell screening model, human TLR2and co-receptors CD14, TLR1and TLR6plasmids and NF-κB promoter-driven luciferase reporter plasmids were co-transfected into Human Embryonic Kidney cells (HEK293). Single clones were then isolated and characterized. Using this screening system, human TLR2-binding peptide C8and Pep2were obtained from the Ph.D.TM-7Phage Display Peptide Library. The binding characteristic of C8and Pep2with human TLR2was evaluated by ELISA, flow cytometry and immunofluorescence. The NF-κB luciferase activity assay showed that C8could activate the TLR2/TLR1signaling pathway. The immunefluorence assay revealed that Pep2could penetrate into TLR2expressing cells. In conclusion, we have successfully established the TLR2receptor-based cell screening system and new TLR2-binding peptides were identified through this system.2. Targeting leukemia and lymphoma with a proapoptotic peptide conjugated to a toll-like receptor2-mediated cell-penetrating peptide.Cell-penetrating peptides provide a unique platform to create a new generation of cancer therapeutics with enhanced efficacy and diminished toxicity. In this study, enhanced expression of toll like receptor2(TLR2) was observed in acute myeloid leukemia (AML) cells. Screening of a phage display peptide library using Bio-panning and Rapid Analysis of Selective Interactive Ligands (BRASIL) identified a TLR2-binding cell penetrating peptide motif, Pep2. We show that Pep2targeted and penetrated into leukemia cells in a TLR2-dependent manner. Moreover, a synthetic, chimeric peptide composed of the TLR2-binding motif linked to a programmed cell death-inducing sequence, D(KLAKLAK)2, induced apoptosis in AML cells with high TLR2expression (TLR2high) but not in chronic myeloid leukemia (CML) cells with low TLR2expression (TLR2low). The anti-leukemia activity of this chimeric peptide was confirmed in leukemia patient samples and an animal model of myeloid leukemia, as the development of leukemia was significantly delayed in mice with TLR2high AML compared to TLR2low CML NOD/SCID mice. TUNEL assays on bone marrow tissue slices revealed that the chimerical peptide induced leukemia cell apoptosis in a TLR2-dependent manner. Together, our findings indicate that TLR2is a potential therapeutic target for the prevention and treatment of AML, and the prototype, Pep2-D(KLAKLAK)2, is a promising drug candidate in this setting.3. TRB3connects autophagy to tumorigenesis and progression.Cancer initiation and progression are associated with autophogy with unclear mechanism. Here we report that silencing of TRB3not only restored suppressed autophagic flux and promoted autophagy-associated cells death, but also attenuated tumor growth and metastasis. However, isotopic expression of TRB3enhanced the susceptibility to tumor development. Notably, TRB3physically interacted with p62to interfere with binding of p62to LC3and ubiquitinated proteins, resulting in an inhibition of autophagy-dependent ubiquitination degradation and accumulation of cancer-promoting factors. Treatment of animals with peptides corresponding to α-helix peptide of p62binding to TRB3attenuated tumor growth and metastasis. Our results demonstrate that TRB3connects autophogy to cancer development and progression. Targeting the TRB3/p62interaction is a therapeutic strategy against cancer progression.