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

Project Sponsor/Mentor:	Donald Bottaro
Title:	None Selected
Address:	Bldg 10 CRC, Rm 2-3961 10 Center Dr MSC 1107 Bethesda, MD 20895
Telephone:	301-402-6499
Fax:	301-402-0922
Email:	dbottaro@helix.nih.gov
Sponsoring Laboratory/Branch:	Urologic Oncology Barnch

Project Title:	Targeting Oncogenic Met Signaling in Cancer
Target(s) of Interest:	Hepatocyte Growth Factor, Met

Project Synopsis:
The biological responses to HGF begin when it binds with high affinity binding to its cell surface receptor, Met, a transmembrane tyrosine kinase (TK). Upon binding, several tyrosines within the Met intracellular domain become phosphorylated, some of which stabilize the active conformation of the kinase and some of which form a multisubstrate binding site for several critical intracellular signal transducers. Thus, antagonism of HGF/Met interaction or inhibition of Met TK catalytic activity should effectively inhibit pathway activation and oncogenic signaling. We have developed assays to measure HGF-Met binding and Met TK activation which provide a basis for identifying antagonists of these events. These assays utilize a state-of-the-art detection technology called electrochemilumescence, which provides improved sensitivity and dynamic range over conventional enzyme-linked detection methods, and each assay is amenable to high-throughput screening of compound libraries. In collaboration with Dr. Barry OKeefe of the NCI Molecular Targets Development Program (MTDP), we will screen a NCI natural products library to identify antagonists of HGF/Met interaction. The natural products library contains ~145,000 extracts collected from all over the world. It includes aqueous and organic extracts from marine invertebrates, microorganisms and plants. Species selected for collection are preferentially endemic to their host countries, which contributes to uniformity of composition and continued availability. Each extract contains thousands of compounds; MTDP scientists have more than 15 years of experience in isolating and characterizing active compounds from natural product extracts. In general, screening natural products libraries is extremely powerful and profoundly relevant to the identification of anti-cancer drug candidates: over the past 25 years, 60-70% of clinically relevant anti-cancer agents were derived from natural product lead structures (Newman et al., J. Nat. Prod, 66:1022-37, 2003). Screening a natural products library is ideally suited to our assay of HGF/c-Met binding for the following key reasons: (1) our current knowledge of HGF/Met interaction cannot yet facilitate the structure-based development of small synthetic binding antagonists, thus complex natural product screening has the greatest potential to identify biologically relevant, readily available binding antagonists; (2) screening is relatively high throughput, and successive rounds of target identification rely on experts in HGF/Met interaction and Molecular Targets Development working together, often in parallel rather than in series; and (3) because HGF/Met interaction occurs outside of target cells, candidates identified in our in vitro screen are unlikely to be rejected at later stages of development for complex issues such as intracellular access. Much evidence suggests that selective TK inhibition is another viable therapeutic strategy for the treatment of several human cancers. In collaboration with Dr. Marc Nicklaus of the NCI Laboratory of Medicinal Chemistry, a virtual in silico screen of the 13.5 million compounds in the ChemNavigator Chemical Library for potential Met TK inhibitors was performed. On the basis of published structural information, a subset of approximately 600,000 compounds was used for molecular docking to reduce this subset to a final set of 175 molecules for biological testing. Four lead compounds with micromolar or submicromolar affinity were identified after biological testing; these were further refined using rational design approaches in collaboration with Drs. Nicklaus and Dr. Terrence Burke of the NCI Laboratory of Medicinal Chemistry (Peach et al., J Med Chem, 52:943-951, 2009). Current preclinical development of these leads will assess their impact on intact cells and in animal models, with iterative design improvements to improve potency and target selectivity. An important feature of our assay for Met activation in cultured cells will be the inclusion of tumor cell lines derived from patients treated in the UOB as well as cell lines engineered to express mutant Met forms. This is a unique approach that builds on the knowledge base of existing Met inhibitors to develop potent compounds with selectivity for Met in combination with other oncogenically-relevant intracellular targets.
Fellow Research Plan:
The trainee will participate in the screening of HGF/Met antagonists, as described in above and, if amenable, the preclinical development of Met TK domain antagonists. The NCI natural products library contains ~145,000 extracts collected from all over the world and is a unique resource for drug screening and development. The Fellow will also receive training from MTDP scientists, who have more than 15 years of experience in isolating and characterizing active compounds from natural product extracts. The trainee will learn electrochemiluminescent assay technology and the basis to improve the scope, efficiency and accuracy of these measurements. Subsequent characterization of lead compounds will include testing with robust assays in use in our lab for cellular activities such as motility, proliferation and matrix invasion, as well as established biochemical analyses of signaling pathways by immunoprecipitation and immunoblotting. We anticipate that new HGF/Met antagonists will show distinct patterns of selectivity for other receptor TKs in vitro, and the trainee will gain extensive training in the rational, structure based design of novel compounds, including the incorporation of novel pharmacophores with potential application to other oncogenic TK protein targets.