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Inhibition of Human Vascular NADPH Oxidase by Biocatalytically Derived Oligophenols.

The role of role of superoxide anion (•O2-) in eliciting oxidative stress is becoming clearer and its importance in vascular diseases is becoming firmer. The production of •O2- is catalyzed by a variety of enzymes, including xanthine oxidase, cytochromes P450, lipoxygenase, enzymes in the mitochondrial respiratory chain, and NADPH oxidases. The latter, in particular, have been identified as the major source of •O2- in vascular endothelial cells (VECs). Excessive production of •O2- in VECs leads to increased oxidative stress and endothelial dysfunction. This in turn can result in a diverse array of cardiovascular diseases, including atherosclerosis, hypertension, diabetes, heart failure, stroke, and restenosis. Due to the key role VEC NADPH oxidase appears to play in vascular diseases, identification of selective inhibitors is of great interest. We have turned our attention to an interesting metabolic process found in the body; namely, the ability of peroxidases to catalyze phenol oxidation in the presence of H2O2, which is often found at the site of oxidative stress. Apocynin (4’-hydroxy-3’-methoxyacetophenone) is a particularly interesting phenol that has been used as an inhibitor of NADPH oxidase. While apocynin itself was found to have low activity in vitro metabolism in vivo converts the phenol into active metabolites that inhibit the enzyme.

Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors [1]. An isolated trimerhydroxylatedquinone (IIIHyQ) showed great inhibitory potential on superoxide production (Figure 1). NADPH oxidaseis believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47phox and p22phox. To that end, while apocynin is unable to block the interaction of his-tagged p47phox with a surface immobilized biotinalyted p22phox peptide, the IIIHyQproduct strongly interfere with this interaction [2]. These results provide evidence that peroxidase-catalyzed AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase. Because their low toxicity, natural derived oligophenols may be an alternative in the treatment of cardiovascular diseases related with oxidative stress.

Figure 1. Apocynin oxidation product (AOP) strongly inhibits endothelial cell (EC) NADPH oxidase. Upper left is the structure of the trimer hydroxylated quinone metabolite of apocynin produced by the action of peroxidases. Upper right is the dose response curve against EC NADPH oxidase. Note the trimer hydroxylated quinone gives an IC50 value of 30 nM. Action of the trimer hydroxylated quinone appears to be due to the disruption of the p47phox-p22phox protein-protein interaction, which is shown in the lower right.


Biological Interactions and Self-Assembly.

Work in this area (in collaboration with Peter Tessier) involves developing a microarray-based assay to detect Aβ1-42 self-interactions that regulate Aβ aggregation and to test the sensitivity of these interactions to two known polyphenolic disruptors of Aβ aggregation (tannic acid and EGCG). We are also using Aβ microarrays to evaluate the inhibitory activities and mechanisms of previously identified phenolic compounds with anti-Aβ aggregation activity. In protein conformational disorders ranging from Alzheimer’s to Parkinson’s disease, proteins of unrelated sequence misfold into a similar array of aggregated conformers ranging from small oligomers to large amyloid fibrils. Substantial evidence suggests that small, prefibrillar oligomers are the most toxic species, yet to what extent they can be selectively targeted and remodeled into non-toxic conformers using small molecules is poorly understood. We have evaluated the conformational specificity and remodeling pathways of a diverse panel of aromatic small molecules against mature soluble oligomers of the Aβ42 peptide associated with Alzheimer disease. We find that small molecule antagonists can be grouped into three classes, which we herein define as Class I, II, and III molecules, based on the distinct pathways they utilize to remodel soluble oligomers into multiple conformers with reduced toxicity (Figure 2) [3,4]. Class I molecules remodel soluble oligomers into large, off-pathway aggregates that are non-toxic. Moreover, Class IA molecules also remodel amyloid fibrils into the same off-pathway structures, whereas Class IB molecules fail to remodel fibrils but accelerate aggregation of freshly disaggregated Aβ. In contrast, a Class II molecule converts soluble Aβ oligomers into fibrils, but is inactive against disaggregated and fibrillar Aβ. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, Aβ non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than Aβ soluble oligomers or amyloid fibrils. Our findings reveal that relatively subtle differences in small molecule structure encipher surprisingly large differences in the pathways they employ to remodel Aβ soluble oligomers and related aggregated conformers.



Figure 2. Remodeling of Aβ soluble oligomers and related aggregated conformers.  Class I molecules remodel Aβ soluble oligomers (ThT- and OC-negative, unstructured as judged by circular dichroism, highly toxic, SDS-soluble) into large aggregates that are SDS-resistant, negative for multiple conformational probes (OC and A11 antibodies, and ThT), unstructured, incompetent for templating Aβ monomers, and non-toxic relative to freshly disaggregated Aβ. In contrast, a Class II molecule selectively converts soluble oligomers into fibrils (which are OC- and ThT-positive, β-sheet rich, SDS-insoluble, A11-negative and mildly toxic). Class III molecules convert soluble oligomers into low molecular species (which are OC-, A11-, and ThT-negative, unstructured, disaggregated as judged by AFM, SDS-soluble and non-toxic relative to freshly disaggregated Aβ). Class IA molecules also convert mature fibrils into large offpathway structures, and these molecules are inactive against freshly disaggregated Aβ. Class IB molecules convert Aβ monomers into large off-pathway aggregates, and are inactive against fibrils. Class II molecules are inactive against Aβ monomers and fibrils. Class III molecules remodel fibrils into low molecular weight species, and they are inactive against freshly disaggregated Aβ.


    1. J. Yu, M. Weïwer, R.J. Linhardt, and J.S. Dordick (2008), “The Role of the Methoxyphenol Apocynin, a Vascular NADPH Oxidase Inhibitor, as a Chemopreventative Agent in the Potential Treatment of Cardiovascular Diseases”, Curr. Vasc. Pharmacol. 6, 204-217.
    2. J.M. Mora-Pale, Michel Weïwer, Jingjing Yu, R.J. Linhardt, and J.S. Dordick (2009), "Inhibition of Vascular NADPH Oxidase by Apocynin Derived Oligophenols”, Bioorg. Med. Chem. 17, 5146-5152.
    3. A.R. Ladiwala, J.C. Lin, S.S. Bale, A.M. Marcelino-Cruz, M. Bhattacharya, J.S. Dordick, and P.M. Tessier (2010), “Resveratrol Selectively Remodels Soluble Oligomers and Fibrils of Amyloid A-beta into Off-Pathway Conformers”, J. Biol. Chem. 285, 24228-24237.
    4. A.R. Ladiwala, J.S. Dordick, and P.M. Tessier (2011), “Aromatic Small Molecules Remodel Toxic Soluble Oligomers of Amyloid-beta Through Three Independent Pathways”, J. Biol. Chem. 286, 3209-3218.