In this proposal we plan to develop therapeutic protease agents designed to target the presumed underlying pathogenic mechanism in Alzheimer's disease (AD) - accumulation of amyloid-beta (Ab) peptide - which proved to be a difficult target for small-molecule strategies. Specifically, we wish to exploit a selection-based directed evolution scheme for rapid identification of proteases with high selectivities and catalytic activities against a specific peptide fragment within Ab. Recent evidence suggests that the formation of amyloid fibrils, as well as the cytotoxic soluble aggregates is an equilibrium-controlled process, triggered by accumulation of Ab. Moreover, defects in both biogenesis and clearance mechanisms of Ab have been linked to the pathogenicity of AD. We hypothesize, therefore, that our catalytic agents will be able to rescue the Ab clearance pathway by exhibiting an internally amplifiable activity against the amyloidogenic peptide. Specifically, we expect that the cleavage of monomoric Ab will be able to 1) shift the aggregation equilibrium toward less complex species and 2) generate potent aggregation inhibitors in situ in the form of proteolysis products. We believe, therefore, that the site-selective proteolysis of Ab presents a unique opportunity for the development of a highly effective therapeutic strategy against this debilitating disease and we plan to accomplish the following Specific Aims in developing this approach: ? ? Specific Aim 1: Using a site-specific protease as a starting scaffold, we plan to develop a genetic selection system capable of identifying uniquely active species with nonnative activities against Ab from a vast collection of genetically encoded sequence permutations. ? ? Specific Aim 2: We will confirm anti-Ab activities of the evolved proteases using an authentic amyloidogenic substrate and further characterize these agents, using 1) kinetic analysis for the substrate specificity analysis, 2) biphasic assays for assessment of their deaggregation properties, and 3) cell viability assay for the measure of their cytoprotective propensity. ? ? Specific Aim 3: Finally, we will optimize the pharmacokinetic properties of the anti-Ab proteases by improving their stability to systemic metabolism and reducing their immonogenicity. ? ? The expected outcome of this research will be identification of therapeutic proteolytic agents capable of attacking putative AD pathogens through catalytic degradation of the Ab monomers into non-fibrinogenic and, potentially, anti-amyloid fragments. Our long-term goal is to develop therapeutic strategies, targeting the underlying pathogenic mechanisms in AD. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AG031437-01
Application #
7361795
Study Section
Special Emphasis Panel (ZRG1-MDCN-C (91))
Program Officer
Buckholtz, Neil
Project Start
2008-03-01
Project End
2010-02-28
Budget Start
2008-03-01
Budget End
2009-02-28
Support Year
1
Fiscal Year
2008
Total Cost
$162,031
Indirect Cost
Name
Purdue University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Verhoeven, Kathryn D; Altstadt, Olvia C; Savinov, Sergey N (2012) Intracellular detection and evolution of site-specific proteases using a genetic selection system. Appl Biochem Biotechnol 166:1340-54
Koley, Dipankar; Colón, Olvia C; Savinov, Sergey N (2009) Chemoselective nitration of phenols with tert-butyl nitrite in solution and on solid support. Org Lett 11:4172-5