Marie Foscue Rourk Professor
Department of Chemistry and Biochemistry
University of North Carolina Greensboro
My research group is a computational chemistry group. We are involved in small molecule modeling and design, in receptor modeling and drug-receptor interaction studies, as well as in simulations of small molecules or proteins in lipid bilayers. Our less compute-intensive calculations are carried out on a network of Silicon Graphics computers in-house, while the high demand calculations, such as the lipid simulations, are conducted on a new Linux cluster here at UNCG and at the Pittsburgh Super Computer Center through a grant of computer time from the PACI program. All of our calculations focus on the cannabinoids (CBs).
By definition, cannabinoids are the group of C21 compounds typical of and present in Cannabis sativa L., their carboxylic acids, analogs, and transformation products. The first well characterized cannabinoid was (-)-trans-D9-tetrahydrocannabinol (D9-THC) which was found to be the major psychopharmacologically active component of cannabis. Receptor cloning studies in the early nineties identified two Cannabinoid receptors, CB1 (found primarily in the brain) and CB2 (found primarily in the immune system). The cannabinoid CB1 and CB2 receptors belong to the Class A, rhodopsin-like family of G protein-coupled receptors. Antagonists (ligands that block the receptor) for each receptor sub-type, as well as four structural classes of agonists (ligands that activate the receptors), have now been identified. Cannabinoid agonists have been suggested to have potential therapeutic uses as appetite stimulants, analgesics, anti-emetics, anti-diarrheals, anti-spasmodics, tumor anti-proliferative agents, anti-glaucoma agents and as agents for the treatment of diseases associated with inappropriate retention of aversive memories such as post-traumatic stress disorders and phobias. Cannabinoid CB1 antagonists/inverse agonists have been suggested to have potential therapeutic uses as appetite suppressants and as agents that improve memory.
At present there are two areas of cannabinoid research that are very hot. The first is the area of inverse agonists. These are agents that bind to the receptor and turn it off, producing nearly opposite effects to cannabinoid agonists. For example, CB1 agonists stimulate appetite, but CB1 inverse agonists reduce appetite. Today, nearly every drug company in the US and Europe has an active cannabinoid inverse agonist project. The second high area of interest is the field of endogenous cannabinoids. These are the ligands that are made in our bodies for specific interaction with cannabinoid receptors. Emerging knowledge about the endocannbinoid system in the brain has indicated that this system was designed to suppress neurotransmitter release from vesicle stores in the brain, exerting a “calming” effect on neurotransmission.
Inverse Agonist Project: Supported by the National Institutes of Health (DA03934)
We are interested in the events that must occur at the molecular level to produce agonist effects and inverse agonist effects. This includes interactions that permit binding to one of the CB receptors and the interactions that are responsible for activating or de-activating the receptor. Such very basic knowledge allows one to rationally design drugs for specific effects. To achieve this goal, it is essential that the receptor models we build accurately reflect the state of knowledge of the cannabinoid receptors. We have been very fortunate to have collaborators who are medicinal chemists, phramacologists and molecular biologists. Our collaborations have allowed us to test hypotheses that we have developed concerning both ligand design and receptor structure. Two recent papers of ours have sparked a lot of interest from the pharmaceutical industry. Through modeling and mutation studies, we have been able to identify the key aromatic residues involved in binding the cannabinoid inverse agonist SR141716A (S.D. McAllister et al. J. Med. Chem. 46, 5139-5152 (2003)). Through modeling/ligand design and functional studies, we have been able to pinpoint the region of the SR141716A molecule (the C-3 substituent) that is responsible for its inverse agonism (Hurst et al. Mol. Pharmacol. 45: 1274-1287 (2002)). Our most recent paper (McAllister et al. J. Biol. Chem. 279, 48024-48037 (2004)). identifies the key interaction (F3.36/W6.48 aromatic stack) that must be stabilized by an inverse agonist to produce inverse effects or broken by an agonist to produce agonist effects.
In the past two years, we have undertaken a study aimed at elucidating how endocannabInoids enter the CB1 receptor and how they bind after entering. The endogenous CB ligand, anandamide (AEA) is synthesized from lipid in neurons, acts at the CB1 receptor and then is degraded by fatty acid amide hydrolase, (FAAH), an integral membrane protein whose active site is accessed by AEA via the lipid bilayer. AEA is the ethanolamide of arachidonic acid (20:4, n-6) and as such, is very lipid-like, including possessing a long alkyl tail. The illustration at left here shows four AEA molecules (light blue) in a DOPC bilayer. The hypothesis we are testing is that AEA approaches the CB1 receptor not from the extracellular milieu, but from lipid; interacts with a specific recognition element on the lipid face of CB1; and, then enters CB1 by passing between transmembrane helices (TMHs) 6 and 7. We have identified a structural motif, a bbxbb motif that creates two grooves on TMH 6 of CB1 into which alkyl chains can fit. One of these grooves (V6.43/I6.46) is present on the lipid face of TMH6 in the inactive state of CB1. We have hypothesized that this groove is the initial recognition element for the alkyl tail of AEA approaching CB1 from lipid. Results of our current molecular dynamics (NAMD) simulations of AEA and TMH6 in a DOPC bilayer have shown that the alkyl tail of AEA in lipid is at the correct depth and angle to interact with the V6.43/I6.46 groove. This result is in contrast to results for a low CB1 affinity 20:2, n-6 analog of AEA, which our simulations have shown is not at the correct depth in the membrane to interact with the V6.43/I6.46 groove. Experimental mutation results for the groove mutant (V6.43A/I6.46A) obtained by our collaborators, show that this mutation separates the binding site of AEA from that of other CB1 ligands that lack alkyl tails (WIN55212-2 and SR141716A). This is still very much a work in progress. We have completed a study of how AEA binds once it enters the receptor (Barnett-Norris et al. J. Med. Chem. 45:3549-3659,(2002)). The more challenging aspect of this problem remains to be how AEA gets into the receptor!
SELECTED PUBLICATIONS FROM THE REGGIO LAB
(from a total of 57 Papers, 10 Reviews and 3 Book Chapters)
R. P. Picone, A. D. Khanolkar W. Xu, L. A. Ayotte, G. Thakur, D. P. Hurst, , M. E. Abood, P. H. Reggio, D. J. Fournier and A. Makriyannis. “AM841, a High-Affinity Electrophilic Ligand Interacts Covalently with a Cysteine in Helix Six and Activates the CB1 Cannabinoid Receptor.” Mol. Pharmacol. 68, 1623-1635 (2005).
R. Zhang, D. P. Hurst, P.H. Reggio and Z.-H. Song. “Cysteine 89(2.59) in the Second Transmembrane Domain of Human CB2 Receptor is Exposed in the Ligand Binding Crevice:Evidence of CB2 Deviation from a Rhodopsin Template.” Mol. Pharmacol. 68, 69-83 (2005).
D. L. Lynch and P.H. Reggio. “Molecular Dynamics Simulations of the Endocannabinoid, N-Arachidonoylethanolamine (Anandamide) in a Phospholipid Bilayer: Probing Structure and Dynamics.” J. Med.Chem. 48, 4824-4833 (2005).
D. Lu, G. A. Thakur, Z. Meng, V. Kumar, P. Fan, J. Steed, C. L. Tartal, D. P. Hurst, P. H. Reggio, T. U.C. Jarbe, and A. Makriyannis. “Adamantyl-(-)-D8-Tetrahydrocannabinols: Novel Ligands for CB1 and CB2 Cannabinoid Receptors”, J. Med. Chem. 48, 4576-4585 (2005).
J. Barnett-Norris, D. Lynch and P. H. Reggio. “Lipids, lipid rafts and caveolae: Their importance for signaling and their centrality to the endocannabinoid system.” Life Sci. 77:1625-1639 (2005).
J. W. Huffman, G. Zengin, M.-J. Wu, J. Lu, G. Hynd, K. Steelman, A. Thompson, S. Bushell, C. Tartal, D. P. Hurst, P. H. Reggio, D. Selley, M. P. Cassidy, Jenny L. Wiley and B. R. Martin.”Structure–Activity Relationships for 1- Alkyl-3- (1- naphthoyl)indoles at the CB1 and CB2 Receptors: Steric and Electronic Effects of Naphthoyl Substituents. New Highly Selective CB2 Receptor Ligands.” Bioorg. Med. Chem. 13, 89-112 (2005).