JHU/CCR Fellowship in Molecular Targets and Drug Discovery Technologies Project Details

Project Sponsor/Mentor:	Kyung S. Lee
Title:	Principal Investigator
Address:	9000 Rockville Pike, Building 37 Room 3118, Bethesda, MD 20892
Telephone:	301-496-9635
Fax:	301-496-8419
Email:	kyunglee@mail.nih.gov
Sponsoring Laboratory/Branch:	Laboratory of Metabolism

Project Title:	Development of anti-polo kinase therapeutic agents
Target(s) of Interest:	Polo-like kinase 1 (Plk1)

Project Synopsis:
Polo-like kinases (Plks) belong to a conserved subfamily of Ser/Thr protein kinases and play an essential role in cellular proliferation. Among them, the most highly conserved polo-like kinase 1 (Plk1) is overexpressed in ~80% of human cancers, and its overexpression is closely associated with oncogenesis in a broad spectrum of human cancers. Interference with Plk1 function induces apoptotic cell death in most tumor cells but not in normal cells and reduces tumor growth in mouse xenograft models. Furthermore, recent genome-wide screens designed to identify components critical for the cancer cell viability singled out Plk1 as the only kinase essentially required for activated Ras or inactivated p53-bearing cancer cells, but not the respective isogenic normal cells. Thus, Plk1 is one of the most important anti-cancer drug targets. Here, we propose to isolate and develop Plk1 inhibitors using various reagents and assays developed in our laboratory. The proposed project is directly related to the generation of anti-Plk1 agents for anti-cancer therapy. Over the years, efforts have been made to generate anti-Plk1 inhibitors, resulting in several compounds (BI2536, GSK Compound 1, Cyclapolin 1, DAP81, and TAL) developed to competitively inhibit the kinase activity of Plk1. However, largely because of the structural similarities among the catalytic domains of various protein kinases, these inhibitors inhibit not only Plk1 but also Plk2 and Plk3, and other closely related kinases. Unlike the function of Plk1, Plk2 and Plk3 rather play an important role in checkpoint-mediated cell cycle arrest to ensure genetic stability and prevent oncogenic transformation. Thus, specific inhibition of Plk1, but not Plk2 or Plk3, is one of the most important issues that need to be addressed for the development of cancer cell-specific anti-Plk1 therapeutic agents. A growing body of evidence suggests that a C-terminal non-catalytic domain termed polo-box domain (PBD) is critical for proper subcellular localization and mitotic functions of Plk1. Our recent studies demonstrated that the nature of the Plk1 PBD binding in recognizing its binding partners is distinct from that of the PBDs from Plk2 and Plk3 (Yun SM, et al., Nat. Str. Mol. Biol. 2009 Jul 13. [Epub ahead of print]). These observations suggest that development of novel inhibitors targeting the Plk1 PBD is a promising strategy for selective inhibition of the Plk1 function. Furthermore, only a 5-mer-long phospho-T78 (p-T78) peptide (PLHSpT), derived from a PBD-binding kinetochore component PBIP1, was sufficient to disrupt the interaction between Plk1 PBD and its physiological binding partners, demonstrating that a small molecular weight inhibitor is sufficient to disrupt the PBD-dependent interaction. With our current understanding of the elements critical for determining the Plk1 PBD-binding affinity and specificity, we have developed an ELISA-based assay that allows us to measure the level of interaction between Plk1 PBD and its binding target. Using this assay, we propose to identify and develop potential Plk1 PBD inhibitors from the NCI natural compound repository. The in vitro high throughput screening (HTS) of the natural compounds will be performed in collaboration with Dr. James McMahon laboratory, while all the biological and biochemical assays will be carried out in our laboratory. Additionally, we are in the middle of developing in vivo cell-based assays with the McMahon group to complement the in vitro HTS. For the past ten years, we have accumulated various experimental assays and other related methods that will allow us to test the potency and selectivity of potential PBD inhibitors in a wide variety of cultured cancer cells and mouse tumor models. 1. In vitro PBD binding and in vivo cell-based HTP screenings (in collaboration with Drs. J. McMahon and S. Kurian) We have developed a novel, highly sensitive, PBD-binding ELISA assay by exploiting the interaction between Plk1 PBD and a small p-T78-containing peptide (Kang YH, et al., Mol. Cell. 2006 24:409-422). In collaboration with the Dr. James McMahon group, we are currently adapting this assay for a HTS format to screen NCIs natural product extract repository and structurally diverse synthetic compounds. Compounds that inhibit the p-T78 peptide-PBD interaction will be isolated and subjected to subsequent biochemical and cell biological studies. We have demonstrated that specific inhibition of the PBD-dependent Plk1 localization is sufficient to disrupt the mitotic functions of Plk1 and, as a result, to induce a potent mitotic arrest and apoptotic cell death (Seong YS. J. Biol. Chem. 2002 277:32282-32293: Yun SM, et al., Nat. Str. Mol. Biol. 2009 Jul 13. [Epub ahead of print]). Thus, examination of the capacity of potential PBD inhibitors to induce this effect in cultured cancer cells will be a simple and easy assay that will allow us to quickly assess the activity of the obtained compounds from the HTS. Our ongoing work on the PLHSpT-derived mimetic peptides proves that this assay is a powerful tool. Our results showed that in vivo Plk1 activity rapidly phosphorylates a T78-bearing peptide and binds to the resulting p-T78 epitope (Park JE and KS Lee, unpublished). By taking advantage of this in vivo event, we are currently developing a two-hybrid type of cell-based assay that utilizes the interaction between the in vivo phosphorylated T78-bearing fusion protein and the Plk1 PBD. With this assay in hand, we plan to identify compounds that disrupt the PBD-interaction by measuring the level of lacZ expression. This assay will serve as a complementary tool for the in vitro HTS above. A fellow will have an opportunity to participate in both in vitro HTS and in vivo cell-based assay. 2. Assays to determine the binding affinity, dynamics, and specificity. This step is to prioritize potential PBD inhibitors based on their biophysical properties. For a set of selected compounds, we plan to carry out isothermal titration calorimetry (ITC) analysis to examine the binding nature and affinity of the compound to the Plk1 PBD. As a second complementary experiment, we will perform surface plasmon resonance (SPR) spectroscopy analyses to investigate the biophysical and biochemical properties of the interactions between PBD and the potential binding molecules. The SPR spectroscopy will provide us not only data concerning the binding affinity between the PBD and the potential inhibitors, but also new insights into the binding dynamics (e.g., transition state) that may likely be essential for future experimental designs. Whether the potential PBD inhibitors obtained above are specific to Plk1, but not to Plk2 or Plk3, is an extremely important issue to be addressed. Testing of the specificity will be performed by subjecting the potential inhibitors to in vitro pull-down assays using the lysates expressing Plk1, Plk2, and Plk3. We will also carry out ITC analyses for these compounds using recombinant PBDs from Plk1, Plk2, and Plk3. Previously, we have successfully used both pull-down assays and ITC analyses to characterize minimal PBD-binding peptides (Yun SM, et al., Nat. Str. Mol. Biol. 2009 Jul 13. [Epub ahead of print]). If necessary, modification of the potential PBD inhibitors will be carried out to enhance the affinity and specificity of the compounds toward the Plk1 PBD (in collaboration with Terry Burke group). 3. Test of lead compounds in cultured cells and other experimental systems. Once the above tested PBD inhibitors specifically disrupt Plk1 functions and inhibit cellular proliferation activity of cancer cells, the next step will be to test their in vivo anti-tumor activities using nude mouse xenograft tumor models that we have used successfully (Park JE, et al. Proc. Natl. Acad. Sci. U S A. 2009. 106:1725-1730). Whether the above tested PBD inhibitors possess preclinical evidence of anti-tumor activity in animal tumor models, either as a single agent or in synergistic combinations with other chemotherapeutic agents, will also be examined to further validate these potential PBD inhibitors. The animal model studies could be carried out as a future direction, not necessarily as a part of fellows proposed research.
Fellow Research Plan:
During the course of conducting this project, the fellow will learn about various aspects of mitotic controls, cellular proliferation, and tumorigenesis. He/she will carry out various biochemical and cell biological assays such as in vitro and in vivo HTS, isothermal titration calorimetry, ELISA assays, time-resolved fluorescence assays, in vitro pull-down assays, cell-based assays, and confocal microscopic analyses. The proposed project employs multifaceted approaches to isolate and develop anti-PBD inhibitors. I anticipate that the multidisciplinary trainings that the fellow will receive will allow him/her to be qualified as an independent researcher in the field of biomedical science. In addition, the fellow will participate in our weekly journal clubs, laboratory group meetings, Laboratory of Metabolism bi-weekly seminars, and other related NIH-wide seminars. He/she will also have an opportunity to present his/her work in a national scientific meeting. I have an experience of working together with predoctoral fellows, publishing their work in high impact journals such as Proc. Natl. Acad. Sci. USA and Mol. Cell. Biol.