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

Project Sponsor/Mentor:	Petr Kalab
Title:	Principal Investigator
Address:	NCI, NIH Bldg 37, Room 2050
Telephone:	301-496 1572
Fax:	30-496 8479
Email:	kalab@mail.nih.gov
Sponsoring Laboratory/Branch:	LCMB

Project Title:	Small molecule inhibitors of Ran function
Target(s) of Interest:	Ran, importin alpha 1, RanBP1

Project Synopsis:
The GTPase Ran regulates nucleo-cytoplasmic transport in the interphase, mitotic spindle assembly and the post-mitotic re-formation of the nuclear envelope. Most of such functions of Ran are mediated by Ran-regulated nuclear transport receptors (NTRs) of the importin beta superfamily. Recent published reports indicated that Ran protein levels are strongly increased in many tumors and in cancer derived tissue culture cells. Moreover, although depletion of Ran by RNAi was well tolerated by normal fibroblasts, the same treatment caused death of cancer-derived tissue culture cells. These observations support the idea that the amplification of Ran and certain Ran- and NTR-regulated pathways may be required for the survival and proliferation of cancer cells in vivo. The mechanism of such putative cancer cell dependence on Ran regulation is not known, although the mitotic role of Ran and NTRs in spindle assembly and function appears to be involved. The Ran-regulated spindle assembly factors (SAFs) include many proteins whose genes are amplified, mutated or aberrantly expressed in cancer: Aurora A, HURP, hTOG, TACC3, NPM1, survivin, BRCA1, APC (adenomatous polyposis coli; manuscript in preparation). In particular, the Ran- and importin brys- regulated importin alpha 1 acts as an inhibitor of several such cancer-related SAFs such as TPX2, the co-activator of Aurora A, a mitotic kinase with oncogenic properties. More than 30 Aurora A kinase inhibitors are being developed by various pharmaceutical companies as potential cancer therapeutics. We propose developing small molecule compounds that would 1) enhance the endogenous inhibitory effect of importin alpha 1 on Aurora A and compounds that would 2) decrease the endogenous levels of RanGTP in cells. Presumably, both types of compounds would have anti-mitotic effects and could lead to the development of new types of cancer therapeutics. We design HTS specifically to search for small molecule inhibitors of protein-protein complex dissociation, i.e. compounds that would be targeting a transient and presumably extremely unique molecular interface, thus decreasing the chances of off-target effects. The rationale for such strategy is justified by numerous examples of highly successful drugs acting as interfacial inhibitors, such as camptothecins, brefeldin, paclitaxel and many others. Specific aims: 1. Development of HTS assays for the inhibitors of importin 1- cargo dissociation 2. Development of HTS assays for the inhibitors of RanBP1-RanGTP interaction 3. Secondary screens with the identified compounds, including screens with NCI-60 cancer cell panel Aim 1 Inhibitors of importin alpha 1- cargo dissociation Each of the 6 human importin alpha isoforms contains flexible N-terminal importin beta binding domain (IBB) and a C-terminal Armadillo repeat domain where the nuclear localization signal (NLS)- containing cargos are loaded. The IBB domain competes with the NLS, preventing cargo binding, unless the IBB is sequestered in complex with importin beta. RanGTP binding to importin alpha dissociates the IBB from importin beta, causing the NLS cargo release from importin alpha. In complex with importin beta the IBB assumes a rod-like alpha -helical conformation. We envision that small molecule compound inhibitors could bind to the binding interface of IBB and importin beta, disrupting the disassembly of the complex and thus enhancing the inhibitory effect of importin alpha 1 on mitotic SAFs. Because the IBB of each importin alpha is slightly different, it should be possible to develop inhibitors specific to the importin alpha 1-importin beta complex. We developed IBB-based molecular sensors whose fluorescence resonance energy transfer (FRET) signal reports on the Ran-GTP regulated binding of IBB to importin beta. Our preliminary 1536 well plate -based tests showed the suitability of these sensors for qHTS. The first task of the Aim 1 will be to modify the existing assay by the inclusion of RanGTP which would allow us to search for IBB-importin beta dissociation inhibitors. The second task of Aim 1 will be to design cell-based HTS assay for importin alpha 1 inhibitors. For example, this task could be accomplished by creating a cell line expressing a fluorescently tagged TPX2 which is a well studied importin alpha 1 cargo. The absence of mostly nuclear localization of TPX2 could serve as readout of the inhibition of importin alpha 1. Aim 2. Inhibitors of RanGTP-RanBP1 interaction RanGTP bound to NTRs such as importin beta is not accessible to RanGAP unless it is extricated from the complex by one of the proteins containing Ran binding domain (RBD). In the absence of RBD, all RanGTP is expected to remain locked up in non-productive complexes with importins and effectively removed from the system. In humans, one of the two RBD-containing proteins is small cytoplasmic protein RanBP1 whose cellular concentration is actively regulated during cell cycle and decreases during cell senescence. We designed FRET sensor (called YRC) based on RBD of RanBP1 that specifically bind to RanGTP and not to RanGDP and report on such interaction by loss of FRET. The task of the Aim 2 will be to design qHTS-ready assay for inhibitors of the RanGTP-RanBP1 interaction. We envision that compounds with such activity would prevent dissociation of RanGTP from NTRs and therefore effectively reduce the cellular concentration of free RanGTP. In principle, the YRC-based HTS can be designed such that it would identify both the inhibitors of RanBP1-RanGTP interaction as well as inhibitors of RanGAP. Aim 3. Secondary screens Whether the small molecule compound inhibitors will be identified through screening at NCGC or in the cell-based HTS system at NCI (Tom Misteli laboratory), the effectiveness and specificity of the compounds will be assessed by several levels of secondary screens. Such assays will be performed with purified proteins, in Xenopus egg extract system, in tissue culture cells as well as in the NCI-60 panel of cancer cell lines. These will be the first steps in the development of highly specific and active inhibitors.
Fellow Research Plan:
Small molecule inhibitors of Ran function Training plan The training plan is modeled on previous experience with high throughput screen (HTS). In this project, where Dr. Kalab participated as a co-author of the FRET-based biosensor used in the screen, was performed at the University of California, Berkeley, and resulted in the identification of a specific small molecule inhibitor of importin beta which is now being further characterized and optimized. We will start with learning the basic theoretical and practical skills required in the project (outlined below). While gradually increasing the complexity of the tasks, most of the training will then take place while participating in the preparation for HTS and in the secondary screens with identified compound hits. Because our HTS assays utilize FRET-based biosensors, the training will provide introduction to various methods of FRET sensor design and analysis. One of the initial goals will be in the optimization and scaling up of HTS assay that has already been validated in pilot screens. However, the project provides opportunities to design new types of biosensors and HTS-ready assays both in purified protein and in live cell format. There are two levels of the outcome of this training project that we are hoping for: 1) The design of HTS assays that will succeed in obtaining NIH funding for the qHTS in the Molecular Library Small Molecule Repository (MLSMR) at the NIH Chemical Genomics Center (NCGC) 2) Identification of candidate small molecule inhibitors through HTS performed either at NCGC or in cell-based HTS system at NCI The funding for qHTS at NCGC is available through a competitive NIH grant process and it is therefore not possible to predict when this screen will be performed. At the earliest, the outcome of our NIH grant application (R03, September 2009) will be known in December 2008. However, we will have an easier access to high throughput screens via the cell-based screening platform which is located at NCI in Bethesda. Training plan 1. Introduction to structure-based design and production of molecular biosensors suitable for HTS During this step, the fellow will be introduced to the basics of a variety of laboratory techniques that are required for the project: molecular cloning (PCR, recombination-based and ligation based, etc.) expression of recombinant proteins, isolation, purification and biophysical protein characterization and measurements of enzyme activity. We will learn the basics of the rational design of functional FRET sensors and how to express, purify and characterize such sensors using spectroscopic and biochemical techniques. 2. Introduction to cell-based assays In this step, the fellow will learn the basics of tissue cell culture techniques, live cell microscopy (including detection of FRET in live cells). We will also learn how to create and characterize cell lines stably expressing fluorescent reporters. 3. Preparation of HTS for importin alpha 1 inhibitors The fellow will participate in the optimization and scaling up of the existing assay that was previously validated by pilot screens at NCGC. Namely, the task here will be to optimize in the laboratory the concentrations of proteins and enzymes involved in the assay (Ran, RCC1) and the conditions for large scale expression of the FRET sensor (YIC) and of Ran and RCC1. The large scale protein production will be contracted to protein production facility at the SAIC, NCI in Frederick, MD. 4. Development of FRET-based HTS for RanGTP-RanBP1 interaction Starting with the existing functional YRC FRET sensor for S. cerevisiae (yeast) RanBP1, the task will be to design an assay that will be use human RanBP1-based FRET biosensor. This will involve the application of structure-based FRET sensor design. 5. Characterization and optimization of the candidate small molecule inhibitors Here, we will learn how to evaluate the effectiveness and specificity of the compounds identified in the HTS screens by a variety of biochemical and cell biological methods. Structural homologues of the identified compound(s) will be evaluated as well, leading to first steps in the rational optimization of the compounds structure.