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A novel photoactive benzoic acid derivative, 4-azido-2-hydroxybenzoic acid (4-AzHBA), is synthesized and employed as a photoprobe to identify the phenol binding site of UDP-glucuronosyltransferases (UGTs). The combination of photoaffinity labeling, enzymatic digestion, MALDI-TOF (matrix-assisted laser desorption/ioninzation time-of-flight) and LC-MS mass spectrometry are used to determine that Phe(super 90) and Phe(super 93) are directly involved in the catalytic activity of UGT1A10 toward 4-MU (4-methylubelliferone) and pNP (p-nitrophenol).

The binding of the saturated diterpenoid phytanic acid, nuclear receptor RXR activator, docosahexaenoic acid, and bile acid lithocholic acid compete for binding to the 9-cis-RA binding site of the human retinoid X receptor beta protein.

Results point out that Glu83 and E134 amino acid residues are essential for the catalytic activity of human phenol sulfotransferase. These carboxyl residues are localized to active site of the enzyme as shown by the use of carboxyl-specific amino acid modification reagent, Woodwards reagent K.

Research has been conducted on the cellular retinoic acid-binding proteins I and II (CRABP-I and -II), the transport proteins for all-trans-retinoic acid. The identification of the amino acids that comprise the CRABP-I's ligand binding site has been carried out via direct photoaffinity labeling.

Synthetic cannabinoids (SCBs), designer drugs marketed as legal alternatives to marijuana, act as ligands to cannabinoid receptors; however, they have increased binding affinity and potency, resulting in toxicity symptoms such as cardiovascular incidents, seizures, and potentially death. N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3- carboxamide (STS-135) is a third generation SCB. When incubated with hepatocytes, it undergoes oxidation, hydrolysis, and glucuronidation, resulting in 29 metabolites, with monohydroxy STS-135 (M25) and dihydroxy STS-135 (M21) being the predominant metabolites. The enzymes responsible for this oxidative metabolism were unknown. Thus, the aim of this study was to identify the cytochrome P450 (P450s or CYPs) enzymes involved in the oxidative metabolism of STS-135. In this study, STS-135 was incubated with liver, intestinal, and brain microsomes and recombinant P450s to determine the enzymes involved in its metabolism. Metabolite quantification was carried out using ultra-performance liquid chromatography. STS-135 was extensively metabolized in HLMs and HIMs. Screening assays indicated CYP3A4 and CYP3A5 could be responsible for STS-135's oxidation. Through incubations with genotyped HLMs, CYP3A4 was identified as the primary oxidative enzyme. Interestingly, CYP2J2, a P450 isoform expressed in cardiovascular tissues, showed high activity towards the formation of M25 with a Km value of 11.4 μmol/L. Thus, it was concluded that STS-135 was primarily metabolized by CYP3A4 but may have extrahepatic metabolic pathways as well. Upon exposure to STS-135, individuals with low CYP3A4 activity could retain elevated blood concentration, resulting in toxicity. Additionally, CYP2J2 may aid in protecting against STS-135-induced cardiovascular toxicity. [ABSTRACT FROM AUTHOR]

Recreational use of marijuana is associated with few adverse effects, but abuse of synthetic cannabinoids (SCBs) can result in anxiety, psychosis, chest pain, seizures and death. To potentially explain higher toxicity associated with SCB use, we hypothesized that AB-PINACA, a common second generation SCB, exhibits atypical pharmacodynamic properties at CB1 cannabinoid receptors (CB1Rs) and/or a distinct metabolic profile when compared to Δ9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive cannabinoid present in marijuana. Liquid chromatography tandem mass spectrometry (LC/MS) identified AB-PINACA and monohydroxy metabolite(s) as primary phase I metabolites (4OH-AB-PINACA and/or 5OH-AB-PINACA) in human urine and serum obtained from forensic samples. In vitro experiments demonstrated that when compared to Δ9-THC, AB-PINACA exhibits similar affinity for CB1Rs, but greater efficacy for G-protein activation and higher potency for adenylyl cyclase inhibition. Chronic treatment with AB-PINACA also results in greater desensitization of CB1Rs (e.g., tolerance) than Δ9-THC. Importantly, monohydroxy metabolites of AB-PINACA retain affinity and full agonist activity at CB1Rs. Incubation of 4OH-AB-PINACA and 5OH-AB-PINACA with human liver microsomes (HLMs) results in limited glucuronide formation when compared to that of JWH-018-M2, a major monohydroxylated metabolite of the first generation SCB JWH-018. Finally, AB-PINACA and 4OH-AB-PINACA are active in vivo , producing CB1R-mediated hypothermia in mice. Taken collectively, the atypical pharmacodynamic properties of AB-PINACA at CB1Rs relative to Δ9-THC (e.g., higher potency/efficacy and greater production of desensitization), coupled with an unusual metabolic profile (e.g., production of metabolically stable active phase I metabolites) may contribute to the pronounced adverse effects observed with abuse of this SCB compared to marijuana. [ABSTRACT FROM AUTHOR]