Developing a PCSK9-competitive inhibitor as a therapeutic agent for reducing blood cholesterol levels

Elevated levels of low‐density lipoprotein cholesterol (LDL-C) in the blood can lead to atherosclerotic plaques and cardiovascular diseases. Cholesterol homeostasis is highly regulated by LDL-Receptor (LDLR), a transmembrane receptor, that mediates the uptake of LDL-C into the liver and thereby clearing it from the circulation. The hepatic enzyme Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) reduces the number of LDLR molecules available to clear LDL-C by directing LDLR to lysosomal degradation. Once PCSK9 binds LDLR on the cell surface, the PCSK9/LDLR complex moves to the acidic endosome through endocytosis, where PCSK9 binds tightly to LDLR and prevents the receptor from escaping and recycling to the membrane. Herein, we are developing a pH-dependent PCSK9-competitive inhibitor that would serve as a potential therapeutic agent to reduce blood LDL-C levels. In this approach, this inhibitor would competitively bind LDLR and thereby prevent its PCSK9-mediated degradation. The PCSK9-competitive inhibitor design is based on two protein engineering strategies, affinity maturation and pH-switch, to generate a mutated variant with higher binding affinity to LDLR at pH 7.4, and reduced affinity at acidic pH, enabling the receptor to dissociate in the acidic endosome and recycle to the cell surface. Thus far, we have produced an active truncated ΔN-PCSK9ΔC protein and established its binding activity to both recombinant LDLR and cell-expressed LDLR at both physiological and acidic pH. Also, the internalization of ΔN-PCSK9ΔC to hepatic cells was demonstrated following interaction with LDLR on the cell surface. This truncated PCSK9 will be further used as a template for designing a library and screening it for high affinity pH-sensitive ΔN-PCSK9ΔC variants.