Crystallographic, kinetic and cellular studies on natural product inspired inhibitors of quinone reductase 2

Katherine C Jermihov, Purdue University

Abstract

Quinone reductase 2 (QR2) is a cytosolic enzyme capable for activating compounds and rendering them capable of causing DNA damage (i.e. DNA alkylation). QR2 has recently been implicated in neurodegenerative diseases such as Parkinson's Disease and schizophrenia, where patients show significant elevation in cellular levels of QR2. Also, the increased levels of reactive oxygen species (ROS) in these patients has been correlated with increased QR2 levels, as its electron transfer mechanisms activate quinones to generate ROS. Here, we report the identification of novel QR2 inhibitors that can potentially be used as new therapeutics for the treatment or prevention of neurodegenerative diseases or to decrease overall toxicity levels of xenobiotic compounds due to QR2. Furthermore, we utilized non-toxic natural products as starting scaffolds to identify inhibitors of QR2. First, the chemical extraction on blueberries, grapes and red wine was performed to isolate small molecules capable of binding to and inhibiting QR2 activity. Through the use of X-ray crystallographic assisted dereplication (CrystAssist), quercetin and quercetin-sugar derivatives were identified that bind to QR2. The binding of these molecules was verified using HPLC and UF-LC/MS (Ultra filtration), in conjunction with enzyme kinetic studies. The extracts were also tested for their ability to inhibit QR2, and the IC50 of the blueberry extract was approximately 10 µg/mL. Finally, a series of synthetic analogs was evaluated for their ability to inhibit QR2 activity, and a structure-activity relationship was developed that incorporated kinetic data, e.g. IC50 values and X-ray crystallographic data on QR2-inhibitor complexes. To further identify QR2 compounds with the potential to decrease oxidative stress (because of QR2 inhibition), we evaluated a series of synthetic resveratrol analogs. Similar to the quercetin analogs, we correlated their potency (IC 50 values) with their structures in complex with QR2, utilizing X-ray crystallography. In this series, over 80 resveratrol analogs were evaluated for inhibition towards QR2, of which measurable potency was observed in 30 compounds. The range in affinity of the 30 compounds was over 2000-fold (from 84 nM to 175 µM), the largest range in affinity as well as the largest number of compounds evaluated of one series as QR2 inhibitors. The third natural product scaffold explored was a metabolite, ammosamide, generated from the isolate of deep-sea marine extracts that showed potent QR2 inhibitory activity. A series of synthetic analogs of ammoasamide was found to display exceptionally high affinity towards QR2, with several compounds demonstrating IC50 values in the low nanomolar range. Using X-ray crystallography, the binding orientations of these analogs in complex with QR2 was determined and were found to be unique. Subtle substitutions in the ammosamide ring structures resulted in drastically different orientations within the QR2 active site. In general, the highest affinity compounds preferred a particular orientation, however, the third most potent compound in the series (PVN-4-28) flips over the y-axis and rotates + 90° relative to the two most potent inhibitors (PVN-5-59 and PVN-4-28). These correlations indicate that binding orientation is not entirely driving by potency of the compound. Finally, it was demonstrated that the novel QR2 inhibitors (from the ammosamide series) are capable of reversing cellular toxicity induced upon increasing menadione concentrations. Menadione is a quinone-containing compound that is metabolized by QR2. It has been hypothesized that menadione reduction results in radical formation, that with the reaction with molecular oxygen, superoxide is formed and is cytotoxic. Here, the addition of the ammosamide analogs was found to counteract the toxicity of increasing menadione concentrations. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Mesecar, Purdue University.

Subject Area

Biology|Molecular biology|Cellular biology|Biochemistry

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