Cholestenoic acid, an endogenous cholesterol metabolite, is a potent γ-secretase modulator.

BACKGROUND: Amyloid-ß (Aß) 42 has been implicated as the initiating molecule in the pathogenesis of Alzheimer's disease (AD); thus, therapeutic strategies that target Aß42 are of great interest. γ-Secretase modulators (GSMs) are small molecules that selectively decrease Aß42. We have previously reported that many acidic steroids are GSMs with potencies ranging in the low to mid micromolar concentration with 5ß-cholanic acid being the most potent steroid identified GSM with half maximal effective concentration (EC50) of 5.7 µM. RESULTS: We find that the endogenous cholesterol metabolite, 3ß-hydroxy-5-cholestenoic acid (CA), is a steroid GSM with enhanced potency (EC50 of 250 nM) relative to 5ß-cholanic acid. CA i) is found in human plasma at ~100-300 nM concentrations ii) has the typical acidic GSM signature of decreasing Aß42 and increasing Aß38 levels iii) is active in in vitro γ-secretase assay iv) is made in the brain. To test if CA acts as an endogenous GSM, we used Cyp27a1 knockout (Cyp27a1-/-) and Cyp7b1 knockout (Cyp7b1-/-) mice to investigate if manipulation of cholesterol metabolism pathways relevant to CA formation would affect brain Aß42 levels. Our data show that Cyp27a1-/- had increased brain Aß42, whereas Cyp7b1-/- mice had decreased brain Aß42 levels; however, peripheral dosing of up to 100 mg/kg CA did not affect brain Aß levels. Structure-activity relationship (SAR) studies with multiple known and novel CA analogs studies failed to reveal CA analogs with increased potency. CONCLUSION: These data suggest that CA may act as an endogenous GSM within the brain. Although it is conceptually attractive to try and increase the levels of CA in the brain for prevention of AD, our data suggest that this will not be easily accomplished.

The stress response neuropeptide CRF increases amyloid-ß production by regulating γ-secretase activity.

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid-ß (Aß) production. In cells, CRF treatment increases Aß production and triggers CRF receptor 1 (CRFR1) and γ-secretase internalization. Co-immunoprecipitation studies establish that γ-secretase associates with CRFR1; this is mediated by ß-arrestin binding motifs. Additionally, CRFR1 and γ-secretase co-localize in lipid raft fractions, with increased γ-secretase accumulation upon CRF treatment. CRF treatment also increases γ-secretase activity in vitro, revealing a second, receptor-independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ-secretase activity. Unexpectedly, CRFR1 antagonists also increased Aß. These data collectively link CRF to increased Aß through γ-secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aß and in some cases preferentially increase Aß42 via complex effects on γ-secretase.

Steroids as γ-secretase modulators.

Aggregation and accumulation of Aß42 play an initiating role in Alzheimer's disease (AD); thus, selective lowering of Aß42 by γ-secretase modulators (GSMs) remains a promising approach to AD therapy. Based on evidence suggesting that steroids may influence Aß production, we screened 170 steroids at 10 µM for effects on Aß42 secreted from human APP-overexpressing Chinese hamster ovary cells. Many acidic steroids lowered Aß42, whereas many nonacidic steroids actually raised Aß42. Studies on the more potent compounds showed that Aß42-lowering steroids were bonafide GSMs and Aß42-raising steroids were inverse GSMs. The most potent steroid GSM identified was 5ß-cholanic acid (EC50=5.7 µM; its endogenous analog lithocholic acid was virtually equipotent), and the most potent inverse GSM identified was 4-androsten-3-one-17ß-carboxylic acid ethyl ester (EC50=6.25 µM). In addition, we found that both estrogen and progesterone are weak inverse GSMs with further complex effects on APP processing. These data suggest that certain endogenous steroids may have the potential to act as GSMs and add to the evidence that cholesterol, cholesterol metabolites, and other steroids may play a role in modulating Aß production and thus risk for AD. They also indicate that acidic steroids might serve as potential therapeutic leads for drug optimization/development.

Comment on "ApoE-directed therapeutics rapidly clear ß-amyloid and reverse deficits in AD mouse models".

Cramer et al. (Reports, 23 March 2012, p. 1503; published online 9 February 2012) demonstrates short-term bexarotene treatment clearing preexisting ß-amyloid deposits from the brains of APP/PS1ΔE9 mice with low amyloid burden, providing a rationale for repurposing this anticancer agent as an Alzheimer's disease (AD) therapeutic. Using a nearly identical treatment regimen, we were unable to detect any evidence of drug efficacy despite demonstration of target engagement.

Cyanobacterial peptides as a prototype for the design of potent ß-secretase inhibitors and the development of selective chemical probes for other aspartic proteases.

Inspired by marine cyanobacterial natural products, we synthesized modified peptides with a central statine-core unit, characteristic for aspartic protease inhibition. A series of tasiamide B analogues inhibited BACE1, a therapeutic target in Alzheimer's disease. We probed the stereospecificity of target engagement and determined additional structure-activity relationships with respect to BACE1 and related aspartic proteases, cathepsins D and E. We cocrystallized selected inhibitors with BACE1 to reveal the structural basis for the activity. Hybrid molecules that combine features of tasiamide B and an isophthalic acid moiety-containing sulfonamide showed nanomolar cellular activity. Compounds were screened in a series of rigorous complementary cell-based assays. We measured secreted APP ectodomain (sAPPß), membrane bound carboxyl terminal fragment (CTF), levels of ß-amyloid (Aß) peptides and selectivity for ß-secretase (BACE1) over γ-secretase. Prioritized compounds showed reasonable stability in vitro and in vivo, and our most potent inhibitor showed efficacy in reducing Aß levels in the rodent brain.