The research team has created a mimetic of the last protein in the pathway, HSP20, which causes relaxation in the same way as the natural protein. This research, published May 8 in the online version of The FASEB Journal, is a major step in the development of a drug that promotes blood vessel relaxation.
The signaling pathway that causes relaxation in smooth muscle cells involves many different proteins, but the last step is the addition of a phosphate group, or phosphorylation, of the protein HSP20, which actually effects relaxation.
Other groups have developed molecules, such as the active ingredient in Viagra, that affect earlier steps in this pathway. But if a problem occurs in later steps, these compounds are ineffective.
"You've got all those signaling pathways, but, boom, you can bypass them by putting in a mimetic of the protein that's the effector molecule," said primary investigator Colleen Brophy, research professor of bioengineering at ASU, director of the Center for Protein and Peptide Pharmaceuticals in the Arizona Biodesign Institute, and chief of vascular surgery at the Carl T. Hayden Veterans Affairs Medical Center.
The HSP20 mimetic developed at ASU consists of a 13 amino acid stretch of the protein attached to a protein transduction domain, a peptide that allows the mimetic to enter cells. The HSP20 portion of the mimetic includes a phosphate group attached to the same amino acid as in the active version of natural HSP20.
Brophy and colleagues measured the contraction of thin rings of smooth muscle from the coronary arteries of pigs with a force transducer. They pre-contracted the muscles by adding the hormone serotonin, then added either their HSP20 mimetic, a scrambled version of the HSP20 mimetic, or papaverine, a compound known to relax muscles by acting earlier in the signal pathway.
The HSP20 mimetic caused the rings of muscle to relax in a dose-dependent fashion, as did papaverine. The scrambled mimetic did not cause any change.
Furthermore, in collaboration with Intrinsic Bioprobes, Inc. of Tempe, Ariz., the researchers used a mass spectrometer to look at the natural HSP20 proteins in the rings. They found both phosphorylated and non-phosphorylated HSP20 in papaverine-treated muscle, but only non-phosphorylated HSP20 in mimetic-treated muscle. Thus, the mimetic induces relaxation by its own action.
Brophy and colleagues also attached a fluorescent protein to their mimetic so that they could see the location of the mimetic in the rings of muscle and individual muscle cells. They found that it was evenly distributed, with a transduction efficiency of about 90 percent.
This research is part of an overarching goal to create protein-based pharmaceuticals to treat a variety of diseases. The next step is to take a HSP20 mimetic into stage one clinical trials so that its efficacy and safety in humans can be tested.
"I'm interested in approaching science from a bedside to bench and back to bedside approach," said Brophy, who is also a vascular surgeon. "We hope to be very applied in terms of looking at clinically relevant problems for which there's an unmet need, then developing experimental approaches to solving these problems, and then, based on experimental discoveries, engineer molecules that can be used to treat human disease."
This research will be published in the print version of The FASEB Journal in July.
Journal
The FASEB Journal