Molecular Determinants for Cannabinoid Activity: Refinement of a Molecular Reactivity Template

The conformation of the active cannabinoid Δ9-THC (I) as detd. by mol. reactivity characteristics was compared to that of the inactive compd. O-methyl-Δ9-THC (II). II was not able to mimic the conformation II of I in which the lone pairs of the phenol O are directed away from ring A. The relation of these results to the biol. activity of I is discussed. Other aspects of the conformation of cannabinoids are reviewed in relation to their biol. activity

Structural Insights from Recent CB1 X-Ray Crystal Structures

Over the past 2 years, X-ray crystal structures of the antagonist- and agonist-bound CB1 receptor have been reported. Such structures are expected to accelerate progress in the understanding of CB1 and should provide an exceptional starting point for structure-based drug discovery. This chapter examines the consistency of these X-ray structures with the CB1 experimental literature, including mutation, NMR and covalent labeling studies. These comparisons reveal discrepancies between this literature and the TMH1-2-3 region of each CB1 crystal structure. The chapter also examines crystal packing issues with each X-ray structure and shows that the discrepancies with the experimental literature can be attributed to crystal packing problems that force the N-terminus deep in the binding pocket of the two inactive state structures and force TMH2 to bend at G2.53/S2.54 and invade the binding pocket in the activated state structure. Revision is advisable before these structures are used for structure-based drug discovery

Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture

We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels. The cells were cultured for 1 d in serum-containing medium and then for up to 25 d in chemically defined medium. In some experiments, epithelial monolayers on gels were mechanically freed from the dishes on day 2 or 5. These gels retracted and formed floating collagen gels. On attached collagen gels, flat monolayers of a single cell type developed within a few days. The cells synthesized DNA until the achievement of confluence but did not accumulate milk proteins. No morphological changes were induced by prolactin (PRL). On floating gels, two cell types appeared in the absence of cell proliferation. The cells in direct contact with the medium became cuboidal and developed intracellular organelles typical of secretory cells. PRL-induced lipogenesis, resulting in large fat droplets filling the apical cytoplasm and accumulation of casein and α-lactalbumin in vesicles surrounding the fat droplets. We detected tranferrin in the presence or absence of PRL intracellularly in small vesicles but also in the collagen matrix in contact with the cell layer. The second cell type, rich in microfilaments and reminiscent of the myoepithelial cells, was situated between the secretory cell layer and the collagen matrix. In embedding gels, the cells formed hollow ductlike structures, which grew continuously in size. Secretory cells formed typical lumina distended by secretory products. We found few microfilament-rich cells in contact with the collagen gels. Storage and secretion of fat, caseins and alpha-lactalbumin required the presence of PRL, whereas the accumulation and vectorial discharge of transferrin was prolactin independent. There was no differentiation gradient between the tip and the cent of the outgrowth, since DNA synthesis and milk protein storage were random along the tubular structures. These results indicate that establishment of functional polarity and induction of cytodifferentiation are influenced by the nature of the interaction of the cells with the collagen structure. The morphological differentiation in turn plays an important role in the synthesis, storage, and secretion of fat and milk proteins

Accelerating Membrane Simulations with Hydrogen Mass Repartitioning

The time step of atomistic molecular dynamics (MD) simulations is determined by the fastest motions in the system and is typically limited to 2 fs. An increasingly popular approach is to increase the mass of the hydrogen atoms to ~3 amu and decrease the mass of the parent atom by an equivalent amount. This approach, known as hydrogen-mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simulation stability. While HMR has been applied in many published studies to date, it has not been extensively tested for membrane-containing systems. Here, we compare the results of simulations of a variety of membranes and membrane–protein systems run using a 2 fs time step and a 4 fs time step with HMR. For pure membrane systems, we find almost no difference in structural properties, such as area-per-lipid, electron density profiles, and order parameters, although there are differences in kinetic properties such as the diffusion constant. Conductance through a porin in an applied field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane mixing show very little dependence on HMR and the time step. We also tested a 9 Å cutoff as compared to the standard CHARMM cutoff of 12 Å, finding significant deviations in many properties tested. We conclude that HMR is a valid approach for membrane systems, but a 9 Å cutoff is not

Cannabinoid ligands targeting TRP channels

Transient receptor potential (TRP) channels are a group of membrane proteins involved in the transduction of a plethora of chemical and physical stimuli. These channels modulate ion entry, mediating a variety of neural signaling processes implicated in the sensation of temperature, pressure, and pH, as well as smell, taste, vision, and pain perception. Many diseases involve TRP channel dysfunction, including neuropathic pain, inflammation, and respiratory disorders. In the pursuit of new treatments for these disorders, it was discovered that cannabinoids can modulate a certain subset of TRP channels. The TRP vanilloid (TRPV), TRP ankyrin (TRPA), and TRP melastatin (TRPM) subfamilies were all found to contain channels that can be modulated by several endogenous, phytogenic, and synthetic cannabinoids. To date, six TRP channels from the three subfamilies mentioned above have been reported to mediate cannabinoid activity: TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, and TRPM8. The increasing data regarding cannabinoid interactions with these receptors has prompted some researchers to consider these TRP channels to be “ionotropic cannabinoid receptors.” Although CB1 and CB2 are considered to be the canonical cannabinoid receptors, there is significant overlap between cannabinoids and ligands of TRP receptors. The first endogenous agonist of TRPV1 to be discovered was the endocannabinoid, anandamide (AEA). Similarly, N-arachidonyl dopamine (NADA) and AEA were the first endogenous TRPM8 antagonists discovered. Additionally, ?9-tetrahydrocannabinol (?9-THC), the most abundant psychotropic compound in cannabis, acts most potently at TRPV2, moderately modulates TRPV3, TRPV4, TRPA1, and TRPM8, though ?9-THC is not reported to modulate TRPV1. Moreover, TRP receptors may modulate effects of synthetic cannabinoids used in research. One common research tool is WIN55,212-2, a CB1 agonist that also exerts analgesic effects by desensitizing TRPA1 and TRPV1. In this review article, we aim to provide an overview and classification of the cannabinoid ligands that have been reported to modulate TRP channels and their therapeutic potential

Towards a better understanding of the cannabinoid-related orphan receptors GPR3, GPR6, and GPR12

GPR3, GPR6, and GPR12 are three orphan receptors that belong to the Class A family of G-protein-coupled receptors (GPCRs). These GPCRs share over 60% of sequence similarity among them. Because of their close phylogenetic relationship, GPR3, GPR6, and GPR12 share a high percentage of homology with other lipid receptors such as the lysophospholipid and the cannabinoid receptors. On the basis of sequence similarities at key structural motifs, these orphan receptors have been related to the cannabinoid family. However, further experimental data are required to confirm this association. GPR3, GPR6, and GPR12 are predominantly expressed in mammalian brain. Their high constitutive activation of adenylyl cyclase triggers increases in cAMP levels similar in amplitude to fully activated GPCRs. This feature defines their physiological role under certain pathological conditions. In this review, we aim to summarize the knowledge attained so far on the understanding of these receptors. Expression patterns, pharmacology, physiopathological relevance, and molecules targeting GPR3, GPR6, and GPR12 will be analyzed herein. Interestingly, certain cannabinoid ligands have been reported to modulate these orphan receptors. The current debate about sphingolipids as putative endogenous ligands will also be addressed. A special focus will be on their potential role in the brain, particularly under neurological conditions such as Parkinson or Alzheimer’s disease. Reported physiological roles outside the central nervous system will also be covered. This critical overview may contribute to a further comprehension of the physiopathological role of these orphan GPCRs, hopefully attracting more research towards a future therapeutic exploitation of these promising targets

GPCRs Moonlighting as Scramblases: Mechanism Revealed

Phospholipids can undergo transverse diffusion, changing leaflets in the bilayer via translocase or scramblase activity. In this issue of Structure, Morra et al. (2018) provide insight into the mechanism used by one scramblase, opsin, based on large-scale ensemble atomistic molecular dynamics simulations. Results support a proposed “credit card reader” model

CBD: A New Hope?

The nonpsychoactive phytocannabinoid, CBD, was recently approved by the Food and Drug Administration for the treatment of children with drug-resistant epilepsy. This milestone opens new avenues for cannabinoid research. In this Viewpoint, we provide an overview of recent progress in the field highlighting molecular insights into CBD’s mechanism of action, as well as its therapeutic potential