Felicia Etzkorn, associate professor of chemistry, will report on her group's achievements in presentations at the 228th American Chemical Society (ACS) National Meeting in Philadelphia August 22-26.
Pin1 regulates the protein Cdc25, which initiates mitosis or cell division. Without Pin1, the cell enters programmed cell death -- which is good if it's a cancer cell.
"It makes sense to inhibit the Pin1 enzyme," said Etzkorn.
Protein shape determines biological function. Pin1 works by changing a peptide bond within Cdc25 from the trans shape (a flattened-Z shape) to the cis U-shape and back. This a simple change -- the swing of an arm one way or another to open or close the U – results in a large conformation change in a protein.
The researchers synthesized cis and trans shaped inhibitors and introduced them into cancer cells. These inhibitors mimic part of the Cdc25 protein but do not bend to the will of Pin1. The research team discovered that cis inhibits the Pin1 enzyme's function 23 times better than trans. "This is very exciting because it tells us something fundamental about the mechanism.
The Etzkorn group tested the inhibitors in an ovarian cancer cell line. "That was the cell line that was available, but it should work for many cancers," Etzkorn said. "It would probably be particularly effective against breast cancer and prostate cancer. It seems to relate to secondary sexual characteristics."
However, a lot of work remains to be done to develop a drug, she cautions. "For instance, the inhibitor does not cross cell membranes. We can overcome that in the laboratory in order to study effectiveness, but have not done animal trials."
Etzkorn concludes, "We are moving closer to validating Pin1 as an anti-cancer target. There is now a small community of scientists looking at inhibiting Pin1, but once it is validated as a target, more researchers will step in."
Etzkorn's talk, "Protein mimics inhibit enzymes that regulate the cell cycle," will be presented at the Division of Organic Chemistry's Women in Organic Synthesis symposium at 10:30 a.m., Monday, Aug. 23, in Ballroom A of the Pennsylvania Convention Center.
The poster, "PhosphoSer-cis-Pro isostere inhibits Pin1 23-fold better than the trans-Pro isostere," by Etzkorn, PhD. student Xiaodong Jane Wang, post-doctoral associate Bailing Xu, former undergraduate Freda K. Neiler, and master's candidate Ashley B. Mullins, all of the Virginia Tech Department of Chemistry, will be presented at the Division of Medicinal Chemistry poster session, 6 to 8 p.m., Sunday, Aug. 22, in Hall D of the Pennsylvania Convention Center.
The poster, " Inverse secondary deuterium isotope effect in Pin1 proline isomerization," by Etzkorn, Mullins, and former chemistry laboratory technician Matthew D. Mason, will be presented at the Protein Function, Enzymology, Heme Biochemistry poster session of the Division of Biological Chemistry, noon to 2 p.m., Tuesday, Aug. 24, Halls A and B of the Pennsylvania Convention Center.
Wang, whose master's degree is from NanKai University, Tianjin, China, is doing her Ph.D. research on inhibiting Pin1. Mullins, of Grundy, Va., is doing her master's research on understanding how Pin1 works. She purified the protein for this research. Neiler, of Fairfax, Va., received her undergraduate degree in human nutrition, foods, and exercise from Virginia Tech and enrolled in the School of Pharmacy at the Medical College of Virginia. She did the ovarian cell based assays for the Etzkorn group research. Mason. who made the protein substrates, is now starting the M.D. program at SUNY Upstate Medical University in Syracuse, N.Y.
The research is sponsored by the NIH (grant RO1GN63271). An invention disclosure and manuscript have been submitted.
Contact for more information: Dr. Felicia Etzkorn, fetzkorn@vt.edu or (540) 231-2235. Learn more about her research at www.chem.vt.edu/chem-dept/etzkorn/homepage.htm
Abstracts
ORGN 213 Protein mimics inhibit enzymes that regulate the cell cycle
Felicia A. Etzkorn, Department of Chemistry, Department of Chemistry, Virginia Tech
Our recent work has focused on designed inhibitors of cell cycle regulatory enzymes: Pin1, Chk1 and histone deacetylases (HDAC). All three of these enzymes represent potential anti-cancer targets. Pin1 is a phosphorylation dependent peptidyl-prolyl isomerase (PPIase) that regulates the G2 to M transition. We have synthesized three conformationally locked pSer-Pro substrate analogues and demonstrated 1-100 É M Pin1 inhibition. The cis is 23-fold more potent against Pin1 PPIase activity than the trans alkene isostere, while a structural isomer control inhibits very poorly. Both pentapeptide analogues are competitive inhibitors, indicating that they bind in the active site of Pin1. Preliminary results in cell-based assays show promising anti-cancer cell activity. Chk1 and Chk2 are the DNA damage checkpoint kinases. The Chk Ser/Thr protein kinases regulate the activity of Cdc25C by phosphorylation and translocation in the cell. A bisubstrate ATP-peptide analogue was synthesized as an inhibitor of Chk1 or 2 and tested for inhibitory activity in vitro.
MEDI 107 PhosphoSer-cis-Pro isostere inhibits Pin1 23-fold better than the trans-Pro isostere
Felicia A. Etzkorn, Xiaodong J. Wang, Bailing Xu, Freda K. Neiler, and Ashley B. Mullins. Department of Chemistry, Virginia Tech
The cell cycle regulator, Pin1 (protein interacting with NIMA #1), is an essential peptidyl-prolyl isomerase. As a potential anti-cancer drug target, Pin1 regulates the activity of several cell cycle enzymes, including Cdc25, Wee1 and Plk1. Two stereoisomeric Pin1 substrate analogues were designed and synthesized. The central pSer-cis/trans-Pro core of the Pin1 substrate was replaced by Z- and E-alkene isosteres. Two other alkene isosteres were also synthesized. The protease-coupled assay demonstrated that all four compounds inhibit Pin1. Both 1 and 2 were competitive inhibitors and the cis isostere, 1, was 23 times more potent (Kis = 1.74 ± 0.08 _M) than its trans counterpart, 2 (Kis = 39.8 ± 2.4 _M). This suggests that Pin1 binds cis substrate at the active site more tightly than trans substrate. Inhibition of the A2780 human ovarian cancer cell line by 1 and 2 correlates well with Pin1 inhibition.
BIOL 101 Inverse secondary deuterium isotope effect in Pin1 proline isomerization
Felicia A. Etzkorn, Ashley B. Mullins, and Matthew D. Mason. Department of Chemistry, Virginia Tech
Pin1 is the most recently discovered member of the family of peptidyl-prolyl isomerases. Pin1 regulates mitosis via activation of Cdc25C phosphatase; as such, it is a potential anti-cancer target. The mechanism of proline isomerization by cyclophilin and FKBP is thought to proceed through a twisted amide transition state, and the model was further refined to postulate a hydrogen bond to the incipient pyramidal nitrogen in the transition state. However, the active site Cys113 of Pin1 has been proposed to act as a nucleophile for the isomerization to proceed through a tetrahedral intermediate. We have measured an inverse secondary deuterium isotope effect (kH/kD = 0.87) for proline-d7 labeled substrate as shown in the figure. This indicates that the prolyl nitrogen is transiently sp3 hybridized and supports our hypothesis that Cys113 may act as a hydrogen bond donor rather than a nucleophile.