Down-regulation of PAR1 activity with a pHLIP-based allosteric antagonist induces cancer cell death

Abstract

Even though abnormal expression of G protein-coupled receptors (GPCRs) and of their ligands is observed in many cancer cells of various origins, only a few anti-cancer compounds directly act on their signaling. One promising approach to modulate their activity consists of targeting the receptor cytoplasmic surfaces interacting with the associated G proteins using peptides mimicking the intracellular loops of the receptor. Thus, to be fully effective, the peptide mimics must be selectively targeted to the tumor while sparing healthy tissues, translocated across the cell membrane and stay anchored to the cytoplasmic leaflet of the plasma membrane. Here, we introduce a novel way to selectively target and inhibit the activity of a GPCR in cancer cells under acidic conditions, such as those found in solid tumors. We find that the conjugation of a peptide fragment derived from the third intracellular loop of the Protease Activated Receptor 1 (PAR1) to a peptide that can selectively target tumors solely based on their acidity (pHLIP), produces a construct capable of effectively down-regulating PAR1 activity in a concentration - and pH-dependent manner, and of inducing a potent cytotoxic effect in a panel of cancer cells that is proportional to the relative level of receptor expression at the cell surface. This strategy not only allows for a more selective targeting and specific intracellular delivery than current approaches, but also offers new possibilities for developing novel anti-cancer drugs targeting GPCRs.

Therapeutic Efficacy of a Family of pHLIP–MMAF Conjugates in Cancer Cells and Mouse Models

“…This is consistent with previous observations that pHLIP is minimally toxic to HeLa cells. 11–13 In addition, cell killing does not appear to be due to chemical instability of the linker in cell media either, as no degradation products are observed after normal and low pH treatments with pHLIP-(WT)–MMAF (Figures S3 and S4). It is consistent with our previous observation that the disulfide bond between pHLIP and MMAE is stable in cell media over the time frame of the cell treatment.…”

Targeting PAR1: Now What?

“…Metastases were inhibited by P1pal-7 alone [81]. A tumor-targeted insertion peptide coupled to intracellular loop 3 of PAR1 also blocks breast cancer growth [83]. PAR1 is selectively overexpressed in patients with high grade infiltrating ductal breast carcinoma compared to lower grade disease [84].…”

Applications of pHLIP Technology for Cancer Imaging and Therapy

“…Also, pHLIPs have been proven to deliver peptides to interfere with the biological action of protein receptors or to disrupt mitochondrial cellular membranes [59, 60]. PAR1 is a protein receptor that is overexpressed in many cancer cells.…”

“…PAR1 is a protein receptor that is overexpressed in many cancer cells. A PAR1 protein fragment was shown to be translocated across the cell membrane by a pHLIP, where it interacted with the intracellular domain of the PAR1 receptor, rendering the protein receptor inactive and inducing pH-dependent cytotoxicity [59]. …”