Analysis of the structure and function of endocannabinoid-hydrolyzing enzymes using biophysical and nanomedical techniques

Abstract

Presented here is a systematic structural, and functional study of endocannabinoid metabolizing enzymes, including: a potential biomarker for breast cancer and the development of a novel nanoplatform for studying these membrane proteins. Investigating the impact of phospholipid bilayers on the structure and function of membrane proteins is an essential precursor to developing drugs that target these dynamic systems. The membrane-associated serine hydrolase, monoacylglycerol lipase (MGL), and the membrane-bound serine hydrolase, fatty acid amide hydrolase (FAAH), are well-recognized therapeutic targets that regulate endocannabinoid signaling. In particular, overexpression of MGL in certain tumor cells elevates the levels of pro-tumorigenic signaling lipids, and as such, MGL regulates a fatty acid network that promotes pathogenesis in some cancers. Reported here is the application of phospholipid bilayer nanodiscs that mimic the native cell membrane environment to evaluate the effects of membrane systems on the catalytic properties, and conformational dynamics, of human MGL (hMGL) and rat FAAH (rFAAH). Specifically, hMGL's kinetic properties (apparent maximum velocity [V_MAX] and substrate affinity [K_M]) were enhanced in the presence of anionic and charge-neutral phospholipid bilayer nanodiscs. In order to examine further the effects of modulating the activity of hMGL, a novel nano-medicinal agent (derived from a dynamic class of nano-materials that can be applied to in vitro, in vivo, and clinical applications) was synthesized, and characterized. Nanoparticles exist in a physical state between that of bulk material and a single molecule. In this transitional state, surface properties and quantum-mechanical dynamics can be "tuned" simply by altering particle size and shape. The `fLPA-SPIO@AuNS' particle design proposed here presents a multi-lamellar nanoplatform for imaging, drug delivery, and therapeutic applications that, in this case, target the endocannabinoid system. Another novel imaging motif, proposed here involves probing the mechanism of hMGL inhibition by 5-(4-hydroxyphenyl)-pentanesulfonyl fluoride (AM3506) using biochemical and mass spectrometric (MS) approaches. After hMGL was treated with AM3506, the conversion of sulfonyl serine (Ser¹²²) to dehydroalanine via a -elimination mechanism was observed, and confirmed by tandem MS analysis. Targeting the resultant dehydroalanine hMGL with thiophenol resulted in the conversion of Ser¹²² to S-phenyl-cysteine (addition of 92 Da), which demonstrates a selective approach for serine hydrolase modification at the catalytic serine. This modification confers a new function to in this case- hMGL without genetic manipulation. The results of this work contribute to the understanding of key regulatory pathway involved in breast cancer progression (as well as other disease states) and provides evidence of the feasibility of the development of a novel, pharmacologic intervention toward the diagnosis and treatment of metastatic disease.