Developing LDLR`s High-Affinity Ligand as a Therapeutic and Delivery Agent

The Low-Density Lipoprotein Receptor (LDLR) plays a critical role in cholesterol homeostasis by mediating the uptake of LDL-cholesterol. LDLR is highly involved in hypercholesterolemia and cancer, which offers an opportunity for a selective targeting of these conditions. Our work is focused on Proprotein Convertase Subtilisin/Kexin type-9 (PCSK9), a regulator of LDLR`s levels that promotes the internalization and degradation of the receptor. Disrupting the interaction of PCSK9 with LDLR is a promising strategy to promote LDLR recycling to the cell surface instead of degradation. We are currently in the process of developing a pH-dependent PCSK9-competitive inhibitor that can serve as a potential therapeutic agent for hypercholesterolemia, and as a high-affinity carrier for cargo delivery specifically into LDLR-expressing cancer cells. It will combine two mechanisms: pH-switch and affinity maturation. In this approach we will use a truncated PCSK9, enhance its affinity toward LDLR, and screen variants with high affinity binding at pH 7.4, and no binding at acidic pH ~5.5. Currently we are producing a truncated ΔN-PCSK9ΔC version in Escherichia coli BL21, and testing its ability to compete with full-length PCSK9 for the binding to LDLR in an LDL uptake assay. We expect the addition of overdosed ΔN-PCSK9ΔC to the cultured cells medium to lower the LDL uptake. This result will be followed by a development of the ΔN-PCSK9ΔC mutated version with higher affinity to LDLR. Furthermore, LDLR is overexpressed in different types of cancer cells, therefore it can be exploited as a portal for the uptake of imaging and therapeutic agents. PCSK9 variants with high affinity and specificity towards LDLR will be tested as a potential vehicle for delivery of cargo into cancer cells.