Joseph W. Harding - US grants

Recent work from our laboratory has identified a new angiotensin-like peptide and its accompanying receptor system. This peptide, generated in vitro from 125I-Angiotensin III (AIII) by CNS membrane preparations, accounts for all the angiotensin specific binding activity seen in the brains of several mammalian species including gerbil, African Green monkey, and rabbit. The focus of this proposal is to establish the in vivo relevance of this new brain peptide system. The work described addresses several important questions: 1. Is the AIII derived peptide (A3DP) found endogenously? 2. How do in vivo levels of A3DP compare with those of other known angiotension peptides, specifically AII and AIII? 3. What is the distribution of A3DP throughout the CNS? 4. Is the A3DP found in vivo the same as that isolated from in vitro preparations? Determining whether this system exists endogenously should provide the insight required to develop new and more effective antihypertensive agents.

The purpose of this proposal is to illuminate the role that membrane-bound peptides play in the overall regulation of the brain angiotensin system. The proposal utilizes a multidisciplinary approach to investigate this problem and as such includes biochemical, behavioral, electrophysiological, and immunocytochemical studies. Several specific questions related to the overall theme are addressed. These include the following: 1) What is the active form of brain angiotensin? 2) What are the functional and biochemical characteristics of membrane-bound angiotensinases? 3) What are the biochemical characteristics of solubilized and purified angiotensin receptors and are they associated with peptidase activity? 4) Can modifications of peptidase activity alter the response of angiotensin-dependent effector mechanisms? And 5) When angiotensin degradation is blocked, does brain angiotensin turnover reflect the physiological state of the animal?

The purpose of this proposal is to illuminate the role that membrane-bound peptides play in the overall regulation of the brain angiotensin system. The proposal utilizes a multidisciplinary approach to investigate this problem and as such includes biochemical, behavioral, electrophysiological, and immunocytochemical studies. Several specific questions related to the overall theme are addressed. These include the following: 1) What is the active form of brain angiotensin? 2) What are the functional and biochemical characteristics of membrane-bound angiotensinases? 3) What are the biochemical characteristics of solubilized and purified angiotensin receptors and are they associated with peptidase activity? 4) Can modifications of peptidase activity alter the response of angiotensin-dependent effector mechanisms? And 5) When angiotensin degradation is blocked, does brain angiotensin turnover reflect the physiological state of the animal?

Hardings
0091337

Recently, Dr. Harding's laboratory discovered a family of cell surface proteins called AT4 receptors. These receptors appear to be important regulators of the fine structure and thus, the function of tissues and organs. One such organ where the activation or blockade of AT4 appears to dramatically impact function is the brain. Here it was found that compounds (developed in the Harding laboratory) that bind AT4 receptors can markedly alter learning efficiency, memory consolidation, and recall. These findings correlate with the high concentration of AT4 receptors in the brain areas associated with cognitive function. The goal of this project is to understand the mechanism by which AT4 receptor modulation influences cognitive function. Specifically, the investigators wish to identify critical cellular processes and molecules that participate in AT4 receptor-dependent effects.

The long-term goals of this project are twofold. First, to clarify the basic mechanisms responsible for learning and memory development and to clarify how the AT4 receptor system fits into the big picture. Second, we wish to identify likely processes and molecules that can contribute to cognitive dysfunction in diseases like Alzheimer's disease. A corollary to this second goal is to determine whether drugs directed at AT4 receptors might serve as useful therapeutics in individuals with cognitive disorders.

DESCRIPTION (provided by applicant): Pancreatic cancer is the fourth leading cause of cancer-related mortality in the United States. Effective treatment strategies have been elusive as evidenced by typical survival times after diagnosis of <1 year. Pancreatic cancer is characterized clinically by its aggressive metastatic activity and resistance to typical cytotoxic chemotherapeutic agents. Recent studies have implicated the over activation of two growth factor systems macrophage stimulating protein(MSP)/RON (its receptor) and hepatocyte growth factor(HGF)/c- Met(its receptor) as critical contributors to pancreatic cancer's ability to disseminate rapidly and its refractoriness to standard chemotherapy. Thus, the objectives of this project are to develop small molecule MSP antagonists and/or dual acting MSP/HGF antagonists that target the dimerization/activation domain of MSP and MSP/HGF for use as pancreatic cancer therapeutics. To reach these objectives the following specific aims will be addressed. 1) Establish that Macrophage-Stimulating protein (MSP) dimerizes. Demonstrate that a peptide representing the putative dimerization domain of MSP (KDYVRT) can block dimerization. 2) Demonstrate that KDYVRT can inhibit the ability of MSP to activate its receptor, RON, and downstream targets by monitoring its effects on MSP-dependent RON, Gab1, akt, and ERK phosphorylation in HEK293 cells. Further demonstrate that KDYVRT can inhibit MSP-dependent effects on HEK293 migration and proliferation. 3) Evaluate the potential of KDYVRT and related molecules to act as dual MSP/HGF antagonists by assessing the ability of KDYVRT and related molecules to concomitantly inhibit HGF-dependent c-Met activation and HGF-dependent cellular responses in HEK293 cells. 4) Demonstrate that KDYVRT can inhibit the growth and survival of MSP/RON and HGF/c-Met sensitive BxPC3 human pancreatic cancer cells as assessed by fluorescent cell sorting methods. 5) Demonstrate that KDYVRT can suppress the growth and metastasis of BxPC3-luc2 human pancreatic cancer cells in an orthotopic NOD-SCID model using live in vivo imaging methodologies and evaluate the effect of KDYVRT on RON and c-Met activation within the tumors. And, 6) Assuming that KDYVRT is effective at blocking RON activation, a limited number of prototype peptides and peptidomimetic will be synthesized that have improved pharmacokinetic properties. Success of these feasibility studies will spur the synthesis of new dimerization domain based molecules with improved pharmacokinetic properties and better bioavailability. Continued success of the development program should lead to the identification of a lead molecule(s), which would be expected to enter clinical development.