Hydrophobic CPP/HDO conjugates: a new frontier in oligonucleotide-warheaded PROTAC delivery

Abstract

Proteolysis-targeting chimeras (PROTACs) have emerged as a potent strategy for inducing targeted degradation of proteins, offering promising therapeutic potential to treat diseases such as cancer. However, oligonucleotide-based PROTACs face significant delivery challenges because of their anionic nature and chemical instability. To address these issues, we developed a novel hydrophobic cell-penetrating peptide (CPP) and heteroduplex oligonucleotide (HDO)-conjugated PROTAC, CPP/HDO-PROTAC, to enhance intracellular delivery and degradation efficiency. CPP/HDO-PROTAC was designed to enter the cell through the activity of the conjugated hydrophobic CPP and release decoy oligonucleotide-based PROTACs by RNase H-mediated RNA strand breaks. Our findings demonstrated that CPP/HDO-PROTAC binds to the estrogen receptor α (ERα) with higher affinity than previous constructs, significantly degrades ERα in MCF-7 human breast cancer cells and inhibits cell proliferation at 10 μM. This research highlights the potential of CPP/HDO-PROTAC as a viable method for delivering and activating decoy oligonucleotide-based PROTACs within cells, overcoming the limitations of traditional transfection methods and paving the way for their clinical application.

Fig. 1. (A) Schematic of the CPP/HDO-PROTAC responsible for degrading ERα. (B) Detailed sequence of the CPP/HDO-PROTAC.

Fig. 2. Decoy-warheaded PROTACs and related molecules used in this study.

Scheme 1. Synthesis of (A) LCL-HDO-F and (B) P4-HDO-R.

Fig. 3. Evaluation of HDO cleavage by RNase H. CPP/HDO-PROTAC, LCL-HDO, LCL-HDO-F, P4-HDO-R, LCL-HDOdna and ER(dec)-R were incubated with RNase H for 2 h. Full-length and digested oligonucleotides were resolved on 20% denaturing polyacrylamide gels.

Fig. 4. Degradation of ERα by the synthesized PROTACs. Whole-cell lysates were analyzed by western blotting with the indicated antibodies (representative data are shown). The numbers below the ERα panels represent the ERα/β-actin ratios, normalized by designating the expression using the vehicle control (condition without a PROTAC) as 100%. (a) Degradation of ERα by CPP/HDO-PROTAC. MCF-7 cells were treated for 24 h with the indicated concentrations of CPP/HDO-PROTAC and LCL-HDOdna. (b) Degradation of ERα by LCL-ER(dec). MCF-7 cells were transiently transfected for 24 h with the indicated concentrations of LCL-ER(dec). An LCL-ER(dec) + P4 mixture or P4 alone was added to MCF-7 cells for 24 h.

Fig. 5. Effects of CPP/HDO-PROTAC on the proliferation of ERα-positive breast cancer cells. Growth inhibition of ERα-positive breast cancer cells by CPP/HDO-PROTAC. MCF-7 cells were treated with 1–10 μM of CPP/HDO-PROTAC or LCL-HDOdna for 72 h, and cell proliferation was then evaluated using a cell viability assay. Data represent the mean ± standard deviation (n = 5).

Fig. 6. Fluorescence microscopy images of MCF-7 cells treated with 10 μM decoy for 2 h. Nuclei and endosomes/lysosomes were stained by Hoechst 33342 and LysoTracker Red, respectively. The fluorescent microscopy observations were performed with a 100× objective lens. Scale bar: 20 μm.