May 19, 2004 | WITH VAST LIBRARIES containing more than 1 million small-molecule candidates to fulfill the quest for the next blockbuster drug, some might think it possible to call off the search for other classes of drugs. But for all their virtues, small molecules have their limitations. They typically act only to inhibit proteins. So why not consider larger molecules, even proteins themselves, as viable drugs?
The simple reason is that proteins are too unwieldy to cross a cell's outer membrane, which provides a virtually impermeable barrier to molecules with a molecular weight greater than about 500 daltons. That makes the goal of introducing a therapeutic protein inside a cell virtually a nonstarter, unless one can summon the molecular equivalent of the Klingon cloaking device or a Trojan horse.
Thanks to a remarkable discovery five years ago, however, that solution appears close. In 1988, researchers noted that an HIV protein called TAT has the uncommon ability to penetrate cells, a property imbued by a positively charged, 11-amino-acid fragment known as a protein transduction domain (PTD). But it was not until 1999 that Steven Dowdy, currently at the University of California at San Diego School of Medicine, reported in Science that tagging a variety of molecules and proteins with this benign TAT peptide miraculously facilitates their entry through the negatively charged cell membrane.
At the time, Dowdy likened the result to "the parting of the Red Sea — it's as if the HIV fragment interacts somehow with the [membrane], opens it up, inserts the protein, and then seals it back up." Time of delivery was extremely rapid, ranging from 30 minutes to a few hours for the blood-brain barrier.
In two studies published earlier this year, Dowdy's group has focused on understanding how the TAT peptide facilitates protein transport across the membrane, as well as applying the technology in a potentially clinical context. In February, his group reported in Nature Medicine that TAT enters cells via a process of "lipid raft macropinocytosis." Simply put, the protein cargo is delivered inside the cell in macropinosomes — relatively long-lived cell vesicles. Dissecting the mechanism of cargo entry now enables researchers to enhance transport efficiency further still.
TROJAN TAT: The HIV fragment effectively escorts a variety of cargo inside cells.
Dowdy's group has looked at TAT tagging to introduce fragments of the notorious tumor suppressor protein p53, the "guardian of the genome," in an effort to re-activate the host protein and subdue the uncontrolled growth of cancer cells. The cancer of choice was peritoneal carcinomatosis because, as Dowdy explains, "we wanted to demonstrate this technique on a malignancy that was a hard one to tackle because it would give us a much better justification for moving this treatment forward toward clinical use."
As reported in PLoS Biology, treatment with this peptide in preclinical mouse models not only increased their longevity sixfold (to more than 200 days), but also resulted in some animals free of disease. It is the first demonstration that activation of endogenous p53 by a macromolecule can be effective in preclinical models of terminal human cancer. In addition to p53, this approach could, in principle, be extended to deliver tumor-suppressor molecules and other anti-cancer proteins to a variety of other targets in vivo.
Not surprisingly, the transduction tag approach has aroused keen interest in biotech. In early 2002, Dowdy founded Ansata Therapeutics with $5 million. Dowdy no longer has any formal ties to the company, which is pursuing new classes of drugs against cancer and neurodegenerative diseases, as well as in-licensing "proprietary cargo" in an effort to develop "commercially rewarding therapeutics for unmet medical needs." Meanwhile, CellGate is already in Phase II clinical trials for treating psoriasis with a transducible cyclosporin.
"As for the future," Dowdy says when asked about other forms of cargo he's exploring, it's "siRNAs, siRNAs, siRNAs!"