Mass spectrometry-based proteomics of human cannabinoid receptor 2: covalent cysteine 6.47(257)-ligand interaction affording megagonist receptor activation.

The lack of experimental characterization of the structures and ligand-binding motifs of therapeutic G-protein coupled receptors (GPCRs) hampers rational drug discovery. The human cannabinoid receptor 2 (hCB2R) is a class-A GPCR and promising therapeutic target for small-molecule cannabinergic agonists as medicines. Prior mutational and modeling data constitute provisional evidence that AM-841, a high-affinity classical cannabinoid, interacts with cysteine C6.47(257) in hCB2R transmembrane helix 6 (TMH6) to afford improved hCB2R selectivity and unprecedented agonist potency. We now apply bottom-up mass spectrometry (MS)-based proteomics to define directly the hCB2R-AM-841 interaction at the amino-acid level. Recombinant hCB2R, overexpressed as an N-terminal FLAG-tagged/C-terminal 6His-tagged protein (FLAG-hCB2R-6His) with a baculovirus system, was solubilized and purified by immunochromatography as functional receptor. A multiplex multiple reaction monitoring (MRM)-MS method was developed that allowed us to observe unambiguously all seven discrete TMH peptides in the tryptic digest of purified FLAG-hCB2R-6His and demonstrate that AM-841 modifies hCB2R TMH6 exclusively. High-resolution mass spectra of the TMH6 tryptic peptide obtained by Q-TOF MS/MS analysis demonstrated that AM-841 covalently and selectively modifies hCB2R at TMH6 cysteine C6.47(257). These data demonstrate how integration of MS-based proteomics into a ligand-assisted protein structure (LAPS) experimental paradigm can offer guidance to structure-enabled GPCR agonist design.

Aliphatic Azides as Selective Cysteine Labeling Reagents for Integral Membrane Proteins.

Upon ultraviolet activation, cannabinergic aliphatic azido (N3) ligands covalently label cannabinoid receptors, prominent G-protein-coupled receptor (GPCR) drug targets. We report here the mechanism of covalent attachment to selected substrates of the high-affinity CBR inverse agonist AM1335 and its deuterated analog AM1335(d10), arylpyrazole compounds with an azide moiety at their n-pentyl side chain. To model the receptor interaction, we utilized the human cannabinoid 2 receptor (hCB2R) transmembrane helix 6 (TMH6) peptide and an N-acyl-protected cysteine (NAC). The photochemical reaction products of model substrates with AM1335 and AM1335(d10) were analyzed with tandem electrospray ionization mass spectrometry fragmentation and deuterium exchange mass spectrometry. The nitrene initially formed after photoreaction undergoes rearrangement to an imine which then interacts with the cysteine sulfhydryl group, resulting in ligand attachment. Our results demonstrate that covalent probes carrying aliphatic azides behave more selectively than originally thought and can be used to label protein cysteine residues preferentially.

A biomimetic Schlemm's canal inner wall: A model to study outflow physiology, glaucoma pathology and high-throughput drug screening.

Glaucoma is a disease that damages the optic nerve, frequently leading to blindness. Elevated intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, which is expected to affect 80 million people by 2020, causing bilateral blindness in over 10 million individuals. Because pathological changes to Schlemm's canal (SC) may account for significant resistance to outflow, there is considerable interest in characterizing and evaluating the Schlemm's canal as a target for glaucoma therapeutics. In conventional, two-dimensional culture, human Schlemm's canal (HSC) cells lose spatial, mechanical and biochemical cues, resulting in altered gene expression and cell signaling than observed in vivo, compromising the clinical relevance of data obtained from such systems. Here, we report, for the first time, that 3D culture of HSC cells on microfabricated scaffolds with defined physical and biochemical cues, rescued expression of key HSC markers, VE-cadherin and PECAM1, and mediated pore formation, crucial for the Schlemm's canal regulation of IOP. We demonstrated that following treatment with the glaucopathogenic agent, TGF-ß2, HSC cells undergo an endothelial-mesenchymal transition, which together with the increase in extracellular matrix (ECM) proteins might account for the decrease in outflow facility observed in patients with high TGF-ß2 levels in their aqueous humor. We also demonstrated that unlike 2D cultures, 3D cultures of HSC cells are amenable to gene transfer. Thus, our data imply that 3D culture of HSC cells may be used as a platform to advance our understanding of HSC physiology and pathology and as a model for high-throughput drug and gene screening.

Direct-detected rapid-scan EPR at 250 MHz.

EPR spectra at 250 MHz for a single crystal of lithium phthalocyanine (LiPc) in the absence of oxygen and for a deoxygenated aqueous solution of a Nycomed triarylmethyl (trityl-CD3) radical were obtained at scan rates between 1.3 x 10(3) and 3.4 x 10(5)G/s. These scan rates are rapid relative to the reciprocals of the electron spin relaxation times (LiPc: T1 = 3.5 micros and T2 = 2.5 micros; trityl: T1 = 12 micros and T2 = 11.5 micros) and cause characteristic oscillations in the direct-detected absorption spectra. For a given scan rate, shorter values of T2 and increased inhomogeneous broadening cause less deep oscillations that damp out more quickly than for longer T2. There is excellent agreement between experimental and calculated lineshapes and signal amplitudes as a function of radiofrequency magnetic field (B1) and scan rate. When B1 is adjusted for maximum signal amplitude as a function of scan rate, signal intensity for constant number of scans is enhanced by up to a factor of three relative to slow scans. The number of scans that can be averaged in a defined period of time is proportional to the scan rate, which further enhances signal amplitude per unit time. Longer relaxation times cause the maximum signal intensity to occur at slower scan rates. These experiments provide the first systematic characterization of direct-detected rapid-scan EPR signals.