Session Topic:
Chemistry of Neuro-disorders: Their mechanism, diagnostics and drug development
Speaker:
Taisuke Tomita, The University of Tokyo
Title:-Secretase as a therapeutic target for Alzheimer's disease
Introduction
Alzheimer’s disease (AD) is a progressive dementing neurodegenerative disorder in the elderly characterized pathologically by neuronal cell loss, and the presence of senile plaques and neurofibrillary changes in the brains of affected individuals. Senile plaquesprimarily contain amyloid- peptides (A) that are proteolytically produced from Amyloid-Precursor Protein (APP). Several lines of evidence indicate that A is the primary molecular culprit that underlies the pathogenesis of AD (“amyloid hypothesis”). Thus, the drugs that can prevent production, aggregation and deposition of A are thought to be a promising therapeutic strategy for AD.
APP is sequentially cleaved - and -secretases to generate A. Thus, these secretases are thought to be plausible molecular targets. In fact, several mega-pharmaceutical companies have reported a number of structurally diverse and potent secretase inhibitors so far. Especially we have been focusing on -secretase, because the -secretase cleavage is the final biological step in Aproduction and determines itslength.Longer A species are the primary and dominantly deposited in the AD brain. Moreover, -secretase cleaves not only APP but other membrane proteins within transmembrane domain. Because usually proteases hydrolyze peptide bonds using ionized water, -secretase is an atypical novel proteasewith loose substrate and cleavage site specificities, and a simple inhibition of -secretase activity may cause a significant adverse effect. Thus, understanding the molecular mechanism whereby -secretase cleaves its substrates is an important issue not only for the therapeutics but also exploring new enzymology and cell biology.
Results and Discussion
-Secretase is a high molecular weight membrane protein complex comprised of presenilin, nicastrin, Aph-1 and Pen-2. However, the structural information of -secretase, that is required for the development of inhibitors/modulators based on its molecular mechanism, is totally lacking. In general, the purification and the structural analysis of membrane protein complex such as x-ray crystallographyareintricate, because of its hydrophobic nature. We have succeeded to developin vitroactivity assay system, the large scale reconstitution and purification of -secretase complex in insect cells.Subsequently, we applied a novel structural analysis method, single particle analysis, using electron microscopy with image processing and found that purified -secretase complex is significantly large enzyme, and resembles a flat heart comprised of two arm-like protrusions. In addition, we found that presenilin, the active site subunit of -secretase, forms a hydrophilic pore within lipid bilayer like channels/transporters by the substituted cysteine accessibility method that enables us to analyze the water accessibility of the residues within transmembrane domains. Finally, we utilized inhibitors, that should bind to -secretase and inhibit with unknown mechanism, for functional analysis; we derivatizedknown inhibitors as molecular probes by addition of photoactivatable moieties to identifytarget molecules. This chemical biological approach provides importantinformation about not only the inhibitory mechanism of compounds, but the functional role of the protein domains. Taken together, combination of traditional biochemical/cell biological analyses and novel techniques would shed light on the structure and molecular mechanism of -secretase.
Concluding remarks
During the past 20 years, the molecular details for the pathogenesis of AD have become clear enough to enable us to develop therapeutic strategies. Ais a key molecule in the etiology of AD and interfering with the secretases could be a promising therapeutics. Combined with genetic studies and biochemical/cell biological analyses, now we are convincingthat -secretase, the plausible molecular targets for AD treatment, is an unusual enzyme and a large membrane protein complex embedded within lipid bilayer. Discovery of several -secretase inhibitors and application for chemical biological analyses dramatically enhanced our understanding of the enzymatic characteristics of -secretase. Further extensive efforts in both academic and pharmaceutical laboratories may raise high hopesfor the establishment of promising therapeutics for AD.
References
Morohashi Y, et al. Carboxyl-terminal fragment of Presenilin is the molecular target of a dipeptidic -secretase-specific inhibitor DAPT J Biol Chem281:14670-14676, 2006
Ogura T, et al. Three-dimensional structure of the -secretase complex. Biochem Biophys Res Comm 343:525-534, 2006
Tomita T and Iwatsubo T. -Secretase as a therapeutic target for treatment of Alzheimer's disease.Curr Pharm Des 12:661-670, 2006
Takasugi N, et al. The role of presenilin cofactors in the -secretase complex. Nature 422:438-441, 2003
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