Wednesday, December 7, 2011

Withanolide A, a Lead Compound for the Treatment of Neurodegenerative Disease

The amyloid cascade hypothesis suggests an imbalance between the production and clearance of the peptide amyloid beta (A beta). The accumulation of A beta peptides in the brain leads to formation of the neuritic plaques that are a distinguishing characteristic of Alzheimer’s disease (AD). Indeed, the level A beta plaques in the brains of AD patients correlates with their degree of cognitive impairment. The A beta pathway implicates several potential AD therapeutic targets and opportunities for pharmacological intervention. Modulation of multiple A beta-regulating targets by a single chemical entity, as opposed to the present one-drug-one-target paradigm, is desirable.
The steroid lactone withanolide A (structure shown above), isolated from Winter Cherry (Withania somnifera), also known as Ashwaganda in ayurvedic medicine, has been shown to down-regulate the expression of beta-secretase 1 (BACE1) in a dose-dependent manner. The same molecule also dose-dependently enhances levels of mature ADAM10; the regulation of each of these targets is consistent with non-amyloidogenic processing of amyloid beta precursor protein (AbetaPP) which suggests that withanolide A may promote disease-modifying effects against AD, as opposed to the current symptomatic therapies. To determine the aforementioned protein expression levels, Chan and co-workers treated primary rat cortical neurons with various doses of withanolide A for 24 hours and then conducted western blot analyses of the cell lysates. It should be noted that the effects noted above were observed with relatively high concentrations of drug (5 – 100 uM). 
What is the precise role of BACE1 and ADAM10 in the amyloidogenic processing of AbetaPP? beta Amyloid is generated by sequential cleavage  events from a larger precursor protein (AbetaPP). First, the aspartic acid protease, beta-secretase (BACE1), cleaves AbetaPP in the extracellular region (see Figure below) to produce a secreted soluble fragment and a membrane-bound C-terminal fragment (C99). Subsequent cleavage of C99 by a separate protease, gamma-secretase, leads to the formation of A beta peptides. Hence, it is clear that downregulation of BACE1 would lead to decreased amyloidogenic processing of AbetaPP. A disintegrin and metalloprotease 10 (ADAM10), a type of alpha-secretase, cleaves AbetaPP within the A beta domain and thus precludes generation of intact A beta peptides. Therefore, upregulation of this enzyme promotes non-amyloidogenic AbetaPP processing by decreasing A beta production.
Protease cleavage of amyloid beta precursor protein.
The multilevel complementary activity of withanolide A may prove effective as a pharmacological therapeutic strategy for AD. Currently, the precise molecular mechanism by which withanolide A modulates levels of BACE1 and ADAM10 is not known. Withanolide A has also exhibited the rare ability to promote neurite outgrowth and reverse neuritic atrophy. In brief, cortical neurons were treated with A beta(25-35), an active partial fragment of amyloid beta, for four days to induce atrophy and then with withanolide A (1 uM). At four days after treatment, the cells were immunostained for an axonal marker or for a dendritic marker. Drug treatment induced regeneration of both axons and dendrites, and achieved reconstruction of pre- and postsynapses in the neurons. Moreover, lengths of the axons and dendrites treated with withanolide A were significantly extended relative to those treated with vehicle alone. Thus, it seems that the steroid lactone withanolide A, in addition to its A beta-related activities, also possesses the unique ability to reconstruct neuronal networks. This AD therapeutic lead compound also exhibited oral in vivo efficacy by recovering A beta(25-35)-induced memory deficits in mice. Memory-retention tests in male mice were quantified with an in vivo assay called the Morris water maze test.
In a forthcoming post, I will examine a practical partial synthesis of withanolide A starting from the readily available steroid precursor pregnenolone. The route was disclosed earlier this year in Angewandte Chemie Int. Ed. by the laboratory of Karl Gademann at the University of Basel.

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