In Vivo Photoadduction of Anesthetic Ligands in Mouse Brain Markedly Extends Sedation and Hypnosis.

Photoaffinity ligands are best known as tools used to identify the specific binding sites of drugs to their molecular targets. However, photoaffinity ligands have the potential to further define critical neuroanatomic targets of drug action. In the brains of WT male mice, we demonstrate the feasibility of using photoaffinity ligands in vivo to prolong anesthesia via targeted yet spatially restricted photoadduction of azi-m-propofol (aziPm), a photoreactive analog of the general anesthetic propofol. Systemic administration of aziPm with bilateral near-ultraviolet photoadduction in the rostral pons, at the border of the parabrachial nucleus and locus coeruleus, produced a 20-fold increase in the duration of sedative and hypnotic effects compared with control mice without UV illumination. Photoadduction that missed the parabrachial-coerulean complex also failed to extend the sedative or hypnotic actions of aziPm and was indistinguishable from nonadducted controls. Paralleling the prolonged behavioral and EEG consequences of on target in vivo photoadduction, we conducted electrophysiologic recordings in rostral pontine brain slices. Using neurons within the locus coeruleus to further highlight the cellular consequences of irreversible aziPm binding, we demonstrate transient slowing of spontaneous action potentials with a brief bath application of aziPm that becomes irreversible on photoadduction. Together, these findings suggest that photochemistry-based strategies are a viable new approach for probing CNS physiology and pathophysiology.SIGNIFICANCE STATEMENT Photoaffinity ligands are drugs capable of light-induced irreversible binding, which have unexploited potential to identify the neuroanatomic sites of drug action. We systemically administer a centrally acting anesthetic photoaffinity ligand in mice, conduct localized photoillumination within the brain to covalently adduct the drug at its in vivo sites of action, and successfully enrich irreversible drug binding within a restricted 250 µm radius. When photoadduction encompassed the pontine parabrachial-coerulean complex, anesthetic sedation and hypnosis was prolonged 20-fold, thus illustrating the power of in vivo photochemistry to help unravel neuronal mechanisms of drug action.

Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells.

Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.

Biochemical mechanism of modulation of human P-glycoprotein by stemofoline.

The resistance to chemotherapeutic drugs by cancer cells is considered to be one of the major obstacles for success in the treatment of cancer. A major mechanism underlying this multidrug resistance is the overexpression of P-glycoprotein (P-gp), resulting in insufficient drug delivery to the tumor sites. A previous study has shown that stemofoline, an alkaloid isolated from Stemona burkillii, could enhance the sensitivity of chemotherapeutics in a synergistic fashion. In the present study, we have focused on the effect of stemofoline on the modulation of P-gp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The effects of stemofoline on a radiolabeled drug, [(3)H]-vinblastine, and fluorescent P-gp substrates, rhodamine 123 and calcein-AM accumulation or retention were investigated to confirm this finding. Stemofoline could increase the accumulation or retention of radiolabeled drugs or fluorescent P-gp substrates in a dose-dependent manner. For additional studies on drug-P-gp binding, P-gp ATPase activity was stimulated by stemofoline in a concentration-dependent manner. More evidence was offered that stemofoline inhibits the effect on photoaffinity labeling of P-gp with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner. These data indicate that stemofoline may interact directly with P-gp and inhibit P-gp activity, whereas stemofoline has no effect on P-gp expression. Taken together, the results exhibit that stemofoline possesses an effective MDR modulator, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells.

Structure of UDP-glucuronosyltransferases in membranes.

This chapter presents the most recent experimental approaches to the investigation of UDP-glucuronosyltransferase (UGTs) in membranes. The first topic described is the subcellular localization of UGTs with special emphasis on the association of these proteins with the endoplasmic reticulum (ER). Experimental methods include subfractionation of tissue for microsome preparation, evaluation of the purity of the membrane fraction obtained, and measurement of UGT activity in the presence of detergents. Next, the recently demonstrated formation of UGT homo- and heterodimer formation and its functional relevance is discussed and the appropriate methods used to characterize such interactions are given (radiation inactivation, size exclusion chromatography, immunopurification, cross-linking, two-hybrid system). The structural determinants of UGTs in relation to membrane association, residency, and enzymatic activity are the next topic, supplemented by a description of the appropriate methods, including the design and expression of chimeric proteins, membrane insertion, and subcellular localization by immunofluorescence. Also presented is new information on the structure and function of UGTs obtained by molecular modeling, bioinformatics (sequence alignment), and comparison with selected crystallized glycosyltransferases. Finally, we discuss the important, and still not fully developed, issue of UGT active site architecture and organization within the ER. This is addressed from two perspectives: (1) chemical modification of UGT active sites by amino acid-specific probes and (2) photoaffinity labeling of UGTs. The detailed synthesis of a photoaffinity probe for an aglycon-binding site is provided and the use of this probe and direct photoaffinity labeling with retinoids is discussed. The application of proteomics techniques, including proteolytic digestion and protein sequencing by liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption ionization/time of flight, to the identification of crucial amino acids of the active sites, and subsequent site-directed mutagenesis of identified amino acids, is discussed in detail.

Polypeptide components of oligomeric legumin-like thiamin-binding protein from buckwheat seeds characterized by partial amino acid sequencing and photoaffinity labeling.

Among thiamin-binding proteins that ubiquitously occur in plant seeds, that of common buckwheat became a model of extensive studies of the chemical mechanism of ligand-protein interaction. In this work, the polypeptide components of buckwheat seed thiamin-binding protein (BSTBP) are identified and characterized. We suggest that BSTBP is probably a fraction of major storage 13 S globulin (legumin), has an average molecular mass of 235 kDa and comprises hexamers of 57-kDa and 38-kDa subunits in variable combinations. Each subunit is a pair of disulfide-linked polypeptide chains, 36 kDa plus 24 kDa and two-times 22 kDa, respectively. The N-terminal sequences of 22-kDa and 24-kDa components show strict homology with those reported for "basic subunits" of buckwheat legumin. By photoaffinity labeling of BSTBP with 4-azido-2-nitrobenzoylthiamine, it is shown that the 36-kDa chain plays the major role in thiamin binding, but the other chains may also be variably involved. Putative thiamin-binding fragments are identified and sequenced.

Novel high-affinity photoactivatable antagonists of corticotropin-releasing factor (CRF) photoaffinity labeling studies on CRF receptor, type 1 (CRFR1).

Novel photoactivatable antagonists of human/rat corticotropin-releasing factor (h/rCRF) have been synthesized and characterized. The N-terminal amino acid D-phenylalanine in astressin ¿cyclo(30-33) [D-Phe12, Nle21,38, Glu30, Lys33]h/rCRF-(12-41)¿, a potent CRF peptide antagonist, was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. Additionally, His32 of astressin was substituted by either alanine or tyrosine for specific radioactive labeling with 125I at either His13 or Tyr32, respectively. The photoactivatable CRF antagonists were tested for their ability to displace 125I-labeled Tyr0 ovine CRF ([125I-labeled Tyr0]oCRF) in binding experiments and to inhibit oCRF-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1) or human Y-79 retinoblastoma cells known to carry endogenous functional human CRFR1 (hCRFR1). ATB-cyclo(30-33)[Nle21,38, Glu30, Ala32, Lys33]h/rCRF-(13-41) (compound 1) was found to bind with higher affinity to rat or human CRFR1 when compared with ATB-cyclo(30-33)[Nle21,38, Glu30, Tyr32, Lys33]h/rCRF-(13-41) (compound 2) and exhibited higher inhibition of oCRF-stimulated cAMP accumulation in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK-rCRFR1 cells) or Y-79 cells. A highly glycosylated, 66-kDa protein was identified with SDS/PAGE, when the radioactively iodinated compounds 1 or 2 were covalently linked to rCRFR1. The specificity of the photoactivatable 125I-labeled CRF antagonists was demonstrated with SDS/PAGE by the finding that these analogs could be displaced from the receptor by their corresponding nonlabeled form, but not other unrelated peptides such as vasoactive intestinal peptide. The observed molecular size of the receptor was in agreement with the size of CRFR1 found in rat pituitary (66 kDa), but was significantly larger than the size of CRFR1 found in rat cerebellum and olfactory bulb (53 kDa).