Protein Phosphatase 5 (PP5) is a serine/threonine phosphoprotein phosphatase belonging to the PPP-family. Both cell culture and animal models of tumor development indicate that PP5 overexpression plays a role in the progression of breast
cancer cells. PP5 expression is responsive to both estrogen and hypoxia inducible factor 1 (HIF-1), with PP5 acting to inhibit stress-induced signaling cascades that trigger apoptosis, programed cell death. When apoptosis of a cancer
cell is inhibited, the cancer cell has a survival advantage and may become resistant to cytotoxic chemotherapeutic agents. The foundation of our research plan is the hypothesis that the inhibition of PP5 will facilitate apoptosis in
cancer cells. Norcantharidin, a derivative of the natural toxin cantharidin, acts as a catalytic inhibitor of PP5 and cytotoxic agent in some types of cancer cells. However, it is not selective and inhibits PP1 and PP2A, other phosphatases
in the same family. This project explores three approaches that span three scientific domains to finding a more selective and potent inhibitor of PP5 that will kill cancer cells.
Specific Aims
Our overarching question is, “how can we develop a viable cancer therapy using PP5 as our target?”. Although our solution is to synthesize norcantharidin derivatives that will selectively inhibit PP5 over PP1 and PP2a with even greater
potency, the paths to reach the solution are multifaceted. My mentors, Dr. Honkanen and Dr. Forbes, have worked on this approach for several years by attaching derivatives to the C-5 (fifth carbon) of norcantharidin. However, my involvement
with this project focuses on three new routes. The first, which was started in May 2024, is opening one of norcantharidin’s rings and attaching a substituent to create a new derivative. The significance here is the stereospecific ring-opening
of the anhydride functionality (a carbonyl bound to an oxygen bound to another carbonyl) of norcantharidin. With this technique, we have made advancements toward the goal of synthesizing a more potent and selective inhibitor by exploiting
the de-symmetrization using a class of compounds called Cinchona alkaloids. The enantiotopic-group differentiation has revealed significant insight into the docking of enantiomers in the active site of PP5, offering an opportunity
to tune next- generation derivatives for future investigations into PP5 selectivity and potency. This will be explored further with the assembly and testing of more stereoisomer inhibitor sets.
The second route, which also started in May 2024, explores the use of norcantharidin as the scaffold for a prodrug that will be taken up more readily by cancer cells by applying the aforementioned ring-opening of norcantharidin's anhydride
functionality. By adding folic acid to the carboxyl to form an ester (carbonyl bound to oxygen), norcantharidin no longer acts to inhibit PPP-family phosphatases. Many types of cancers, such as triple-negative breast cancer, overexpress
folic acid transporters, aiding cell proliferation. Thus, the norcantharidin-folic acid prodrug should be taken up more readily by the cancer cells that over-express folic acid uptake mechanisms. Once inside a cell, the ester should
be readily hydrolyzed, liberating the cytotoxic component endothall (the hydrolyzed version of norcantharidin). The compound would not act as an inhibitor until taken up by the cancer cells, making it more selective for cancer cells.
After the assembly of the prodrug in chemistry (Forbes Lab), the assessment of efficacy would be conducted in the College of Medicine (Honkanen Lab) through a series of cell-based (in vitro) dose response assays.
Finally, the third aspect, and the focus of this funding opportunity, features the assembly of bioconjugates (molecules made up of one biomolecule covalently bound to at least one other molecule) of the norcantharidin molecule to realize
the assembly of both a potent and selective inhibitors of PP5 in an even more unique and multidisciplinary way. This will be accomplished by tethering two functionalized derivatives, a sulfhydryl-specific and a PP5-specific derivative,
to a bio-compatible gold nanocluster protected by bidendate N-heterocyclic carbenes (NHC). Once synthesized, the goal would be to test it in vitro to see if it would be a plausible candidate for a challenging and complicated area of
cancer therapy, the Enhanced Permeability and Retention (EPR) effect. 1,2 If so, the intent would be that the size-specific nanoclusters circulate throughout the body and preferentially accumulate at the tumor. The final aspect of
the project will be pursued at the Institute of Transformative BioMolecules (ITbM) at Nagoya University in Japan over the summer of 2026.
Interdisciplinary Research Components (excerpt)
To answer the question of how we can develop a viable cancer therapy drug that targets PP5, this project spans across three domains: biomedical, organic chemical, and materials science research. This project would not be possible without
the collaboration of my mentors, Dr. Honkanen and Dr. Forbes, and their labs in the USA Whiddon College of Medicine and the USA Department of Chemistry. Dr. Honkanen and his collaborator Dr. Swingle are known globally for their work
on protein phosphatases, as Dr. Honkanen is the discoverer of protein phosphatase 5 and inventor of the in vitro assays specific for PP5. 17 Dr. Forbes has been nationally recognized as a Cottrell Scholar in 2000 for his outstanding
mentorship of undergraduate students and contributions to research. He has also been recognized through his appointment as a member of the Science Advisory Council of the Beckman Foundation. Three of his undergraduate students have
been recognized as Goldwater Scholars, two have been recognized as Phi Kappa Phi Marcus Urann Fellows, and one has been recognized as a National Mortar Board Fellow.
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