Hydra-Elastin-like Polypeptides Increase Rapamycin Potency When Targeting Cell Surface GRP78

Abstract

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and doselimiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucoseregulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

Cell surface GRP78: a potential mechanism of therapeutic resistant tumors

“…Additionally, the researchers linked 5FA with L peptide (RLLDTNRPLLPY), a ligand for CS-GRP78, which enhances the uptake of rapamycin through the mTORC1 pathway. These “Hydra-ELPs” have been found to increase the potency of rapamycin and enhance its ability to sensitize cancer cells when targeting the cell surface form of GRP78 [ 78 ]. Polymeric nanoparticles carrying a reactive peptide for HSPA5 and loaded with Tx have been shown to slow down the growth of solid tumor cells in vivo and enhance the apoptosis of these cells when exposed to radiation [ 79 ].…”

Engineered elastin-like polypeptides: An efficient platform for enhanced cancer treatment

“…Avila H prepared an ELPs-based nanocarrier containing rapamycin-binding domains for targeting glucose-regulated protein (csGRP78). The targeted carriers significantly enhanced cellular uptake and reduced mTOR activity by 3-fold compared to free rapamycin ( Avila et al, 2022 ). Ramamurthi D also prepared gemcitabine-conjugated ELPs for ovarian cancer therapy ( Ramamurthi et al, 2022 ).…”

Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents

“…ELP sequences are composed of Val-Pro-Gly-X-Gly pentapeptide repeats, inspired by tropoelastin, where X can be any amino acid except proline. ELPs display a low critical solution temperature (LCST) and undergo a reversible phase separation at their transition temperature. − Moreover, ELPs are responsive to various stimuli, including pH and ionic strength, and these characteristics have been employed to create diverse nanostructures. , This versatility enables ELP fusions with functional proteins, making them suitable for applications in wound healing, drug delivery, and other therapeutic purposes. − Their temperature responsiveness has been employed to create microarchitectures that exhibit LCST phase behavior, leading to the formation of distinct hollow microshell emulsions that can be stabilized through UV cross-linking. In addition, a self-assembling, ELP-oligonucleotide diblock has been synthesized to form thermosensitive stable micellar structures .…”