(−)-CHANA, a Fluorogenic Probe for Detecting Amyloid Binding Alcohol Dehydrogenase HSD10 Activity in Living Cells

Muirhead, Kirsty E. A.; Froemming, Mary K.; Li, Xiaoguang; Musílek, Kamil; Conway, Stuart J.; Sameš, Dalibor; Gunn-Moore, Frank J.

doi:10.1021/cb100199m

Cited by 16 publications

(26 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The toxicity at higher concentrations argues for a proper dosing. The concentration range applied by us is consistent with the IC 50 determined for AG18051 in a previous study [47], [48]. With increasing concentrations of the inhibitor, as expected, estradiol levels were reduced (Fig.…”

Section: Resultssupporting

confidence: 90%

Inhibition of the Mitochondrial Enzyme ABAD Restores the Amyloid-β-Mediated Deregulation of Estradiol

et al. 2011

View full text Add to dashboard Cite

Alzheimer's disease (AD) is a conformational disease that is characterized by amyloid-β (Aβ) deposition in the brain. Aβ exerts its toxicity in part by receptor-mediated interactions that cause down-stream protein misfolding and aggregation, as well as mitochondrial dysfunction. Recent reports indicate that Aβ may also interact directly with intracellular proteins such as the mitochondrial enzyme ABAD (Aβ binding alcohol dehydrogenase) in executing its toxic effects. Mitochondrial dysfunction occurs early in AD, and Aβ's toxicity is in part mediated by inhibition of ABAD as shown previously with an ABAD decoy peptide. Here, we employed AG18051, a novel small ABAD-specific compound inhibitor, to investigate the role of ABAD in Aβ toxicity. Using SH-SY5Y neuroblastoma cells, we found that AG18051 partially blocked the Aβ-ABAD interaction in a pull-down assay while it also prevented the Aβ42-induced down-regulation of ABAD activity, as measured by levels of estradiol, a known hormone and product of ABAD activity. Furthermore, AG18051 is protective against Aβ42 toxicity, as measured by LDH release and MTT absorbance. Specifically, AG18051 reduced Aβ42-induced impairment of mitochondrial respiration and oxidative stress as shown by reduced ROS (reactive oxygen species) levels. Guided by our previous finding of shared aspects of the toxicity of Aβ and human amylin (HA), with the latter forming aggregates in Type 2 diabetes mellitus (T2DM) pancreas, we determined whether AG18051 would also confer protection from HA toxicity. We found that the inhibitor conferred only partial protection from HA toxicity indicating distinct pathomechanisms of the two amyloidogenic agents. Taken together, our results present the inhibition of ABAD by compounds such as AG18051 as a promising therapeutic strategy for the prevention and treatment of AD, and suggest levels of estradiol as a suitable read-out.

show abstract

Section: Resultssupporting

confidence: 90%

Inhibition of the Mitochondrial Enzyme ABAD Restores the Amyloid-β-Mediated Deregulation of Estradiol

et al. 2011

View full text Add to dashboard Cite

show abstract

“…Inhibitory kinetics were determined over a course of Aβ concentrations, and the inhibitor constant (K i ) was determined to be 2.5 μM (Fig. 5B), in agreement with previous reports of HSD10 inhibition using a soluble substrate (Muirhead et al 2010). …”

Section: The Aβ Peptide Inhibits Oxidation Of CL By Hsd10supporting

confidence: 88%

Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases

Boynton

Shimkets

2015

Genes Dev.

View full text Add to dashboard Cite

Myxococcus xanthus development requires CsgA, a member of the short-chain alcohol dehydrogenase (SCAD) family of proteins. We show that CsgA and SocA, a protein that can replace CsgA function in vivo, oxidize the 2 ′ -OH glycerol moiety on cardiolipin and phosphatidylglycerol to produce diacylglycerol (DAG), dihydroxyacetone, and orthophosphate. A lipid extract enriched in DAGs from wild-type cells initiates development and lipid body production in a csgA mutant to bypass the mutational block. This novel phospholipase C-like reaction is widespread. SCADs that prevent neurodegenerative disorders, such as Drosophila Sniffer and human HSD10, oxidize cardiolipin with similar kinetic parameters. HSD10 exhibits a strong preference for cardiolipin with oxidized fatty acids. This activity is inhibited in the presence of the amyloid β peptide. Three HSD10 variants associated with neurodegenerative disorders are inactive with cardiolipin. We suggest that HSD10 protects humans from reactive oxygen species by removing damaged cardiolipin before it induces apoptosis.

show abstract

“…This novel approach has recently been improved upon by the means of synthesis of the individual stereoisomers of CHANA. ( − )-CHANA was found to be selectively metabolized by ABAD, whereas ( + )-CHANA showed a much higher level of background oxidation by other cellular dehydrogenases [57]. Thus, using ( − )-CHANA, we have laid the foundations for a cellbased assay monitoring of ABAD activity.…”

Section: Therapeutic Targets and Development Of Assaysmentioning

confidence: 99%

“…Upon addition of 22 μM Aβ(1-42), HEK-293 cells (human embryonic kidney cells) were shown to exhibit a significant decrease in ( − )-CHANA metabolism of approximately 20%. It is hoped that further development of this system will provide a robust cell-based assay for use in the identification of potential modulators of ABAD function [57].…”

Section: Therapeutic Targets and Development Of Assaysmentioning

confidence: 99%

Mitochondrial β-amyloid in Alzheimer's disease

Borger

Aitken

Muirhead

et al. 2011

Biochemical Society Transactions

View full text Add to dashboard Cite

It is well established that the intracellular accumulation of Aβ (amyloid β-peptide) is associated with AD (Alzheimer's disease) and that this accumulation is toxic to neurons. The precise mechanism by which this toxicity occurs is not well understood; however, identifying the causes of this toxicity is an essential step towards developing treatments for AD. One intracellular location where the accumulation of Aβ can have a major effect is within mitochondria, where mitochondrial proteins have been identified that act as binding sites for Aβ, and when binding occurs, a toxic response results. At one of these identified sites, an enzyme known as ABAD (amyloid-binding alcohol dehydrogenase), we have identified changes in gene expression in the brain cortex, following Aβ accumulation within mitochondria. Specifically, we have identified two proteins that are up-regulated not only in the brains of transgenic animal models of AD but also in those of human sufferers. The increased expression of these proteins demonstrates the complex and counteracting pathways that are activated in AD. Previous studies have identified approximate contact sites between ABAD and Aβ; on basis of these observations, we have shown that by using a modified peptide approach it is possible to reverse the expression of these two proteins in living transgenic animals and also to recover mitochondrial and behavioural deficits. This indicates that the ABAD-Aβ interaction is potentially an interesting target for therapeutic intervention. To explore this further we used a fluorescing substrate mimic to measure the activity of ABAD within living cells, and in addition we have identified chemical fragments that bind to ABAD, using a thermal shift assay.

show abstract

(−)-CHANA, a Fluorogenic Probe for Detecting Amyloid Binding Alcohol Dehydrogenase HSD10 Activity in Living Cells

Cited by 16 publications

References 23 publications

Inhibition of the Mitochondrial Enzyme ABAD Restores the Amyloid-β-Mediated Deregulation of Estradiol

Inhibition of the Mitochondrial Enzyme ABAD Restores the Amyloid-β-Mediated Deregulation of Estradiol

Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases

Mitochondrial β-amyloid in Alzheimer's disease

Contact Info

Product

Resources

About