Faculty Collaboration Database

Endocannabinoid Transport Proteins: Discovery of Tools to Study Sterol Carrier Protein-2. Methods Enzymol 2017;593:99-121

Date

07/29/2017

Pubmed ID

28750817

Pubmed Central ID

PMC6904209

DOI

10.1016/bs.mie.2017.06.017

Scopus ID

2-s2.0-85024124936 (requires institutional sign-in at Scopus site)   24 Citations

Abstract

The endocannabinoid (eCB) neurotransmitter system regulates diverse neurological functions including stress and anxiety, pain, mood, and reward. Understanding the mechanisms underlying eCB regulation is critical for developing targeted pharmacotherapies to treat these and other neurologic disorders. Cellular studies suggest that the arachidonate eCBs, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are substrates for intracellular binding and transport proteins, and several candidate proteins have been identified. Initial evidence from our laboratory indicates that the lipid transport protein, sterol carrier protein 2 (SCP-2), binds to the eCBs and can regulate their cellular concentrations. Here, we present methods for evaluating SCP-2 binding of eCBs and their application to the discovery of the first inhibitor lead molecules. Using a fluorescent probe displacement assay, we found SCP-2 binds the eCBs, AEA (K_i=0.68±0.05μM) and 2-AG (K_i=0.37±0.02μM), with moderate affinity. A series of structurally diverse arachidonate analogues also bind SCP-2 with K_i values between 0.82 and 2.95μM, suggesting a high degree of tolerance for arachidonic acid head group modifications in this region of the protein. We also report initial structure-activity relationships surrounding previously reported inhibitors of Aedis aegypti SCP-2, and the results of an in silico high-throughput screen that identified structurally novel SCP-2 inhibitor leads. The methods and results reported here provide the basis for a robust probe discovery effort to fully elucidate the role of facilitated transport mediated by SCP-2 in eCB regulation and function.

Author List

Hillard CJ, Huang H, Vogt CD, Rodrigues BE, Neumann TS, Sem DS, Schroeder F, Cunningham CW

Author

Cecilia J. Hillard PhD Associate Dean, Center Director, Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Arachidonic Acids
Binding, Competitive
Carrier Proteins
Endocannabinoids
Humans
Ligands
Models, Molecular
Protein Binding