NANO.IL.

Developing a PCSK9-competitive Inhibitor as a Therapeutic and Delivery Agent

Lital Ben-Naim Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel National Institute of Biotechnology, Ben-Gurion University of the Negev, Beer-Sheva, Israel

The Low-Density Lipoprotein Receptor (LDLR) is a transmembrane protein that is highly involved in Hypercholesterolemia and Cancer, and plays a critical role in cholesterol homeostasis by mediating the uptake of LDL-cholesterol. Our work is focused on Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) that binds to LDLR and directs it to lysosomal degradation, which leads to reduced uptake of LDL-cholesterol and its accumulation in the plasma. Degradation of LDLR via interaction with PCSK9 is one of the significant causes for hypercholesterolemia. Preventing the interaction of native PCSK9 to LDLR is a promising strategy to enable LDLR to escape the degradation pathway in low pH, recycle back to the cell membrane and thereby lower circulating LDL-cholesterol levels. We are currently developing a pH-dependent PCSK9-competitive inhibitor that would serve as a potential therapeutic agent for hypercholesterolemia, and as a potential carrier for delivery of cargo specifically into LDLR-over-expressing cancer cells. Our PCSK9 inhibitor design is based on two protein engineering strategies, namely pH-switch and affinity maturation.

Since the C-terminal tail of PCSK9 is required for mediated degradation of LDLR at acidic pH, a truncated version of PCSK9 lacking the C-terminal domain is used in order to prevent PCSK9 binding to LDLR at low pH. Thus far, we produced a stable and active short version of ΔN-PCSK9ΔC-His (Ala53-Ala451) in Escherichia coli, and established its binding activity to LDLR. Moreover, ΔN-PCSK9ΔC-His ability to enter the cells following interaction with the LDLR on cell surface was demonstrated.

This truncated form of PCSK9 will be further used as a template for developing a mutated variant of ΔN-PCSK9ΔC with higher affinity and specificity to LDLR in comparison to the native PCSK9. Furthermore, the ΔN-PCSK9ΔC variant will be tested as a vehicle for delivery of imaging and therapeutic cargo into LDLR--expressing cancer cells.









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