pHLIP, a peptide discovered in the Engelman lab, has promising therapeutic applications

Don Engelman
October 6, 2022

In collaboration with a research team from the University of Rhode Island and the Miriam Hospital in Providence, MB&B professor, Don Engelman, Ph.D, recently published an article in Frontiers in Urology. The article, “Targeting bladder urothelial carcinoma with pHLIP-ICP and inhibition of urothelial cancer cell proliferation by pHLIP-amanitin,” explores a unique property of a peptide derived from bacteriorhodopsin that was initially characterized in the Engelman lab, and expanded upon by Dr. Yana Reshetnyak.

The pHLIP (pH-Low Insertion Peptide) molecule is one of seven peptide helices bound together in the bacteriorhodopsin membrane protein. Seminal research from the Engelman lab showed that one of the moderately hydrophobic helices– what is now known as pHLIP– is reversibly absorbed by cell membranes at normal and high pHs, but at low local pHs (pH 6.0-6.5) pHLIP peptides insert across the cellular membrane to form a stable transmembrane helix. In 2003, Dr. Reshetnyak joined the Engelman lab as a postdoctoral fellow and started working on pHLIP. Since cancer cells are generally abnormally acidic compared to healthy cells due to dysregulated metabolic processes, Dr. Reshetnyak and Dr. Engelman wondered if pHLIP could be used to target cancer cells. Since the initial discovery in 2003, it has been shown that you can attach chemotherapy drugs, immuno-modulating drugs, and even fluorescent molecules to pHLIP. Considering the pH targeting properties of pHLIP, it can be used to deliver these molecules directly, and specifically, to cancer cells to either tether the cargo to the cellular membrane or deliver the cargo through the membrane.

In this new paper, the scientists at the University of Rhode Island and Yale use pHLIP as a molecular delivery system for imaging urothelial cancer cells in patient bladder tissue. The two agents used for imaging are pHLIP-ICG and pHLIP-IR800, two fluorescent NIRF dyes that have spectral properties well suited for clinical imaging. They showed that pHLIP-ICG targets human urothelial carcinoma in ex-vivo human bladder specimens with 98% sensitivity and 100% specificity. pHLIP-ICG is currently being investigated through clinical trials for use in fluorescent-guided surgeries.

The team also tested the therapeutic potential of pHLIP-amanitin, a cytotoxin attached to the peptide’s membrane-inserting end via a cleavable S-S link. Alpha-amanitin is a highly toxic agent which inhibits eukaryotic polymerase II (Pol II) which leads to Pol II degradation and cell death. Selectively delivering this and other agents to cancerous cells has a high potential for therapeutic applications. The results from these studies are very encouraging for the potential use of pHLIP-based agents in cancer treatments. Therapeutic and clinical trials are currently being conducted that use therapeutic pHLIP conjugates as treatments for metastatic cancers.

Learn more about these clinical breakthroughs that spun out of discoveries made in the Engelman lab in the new article in Frontiers in Urology.

By Jake Thrasher