Retinoic acid (RA) is a critical signaling molecule that regulates gene transcription and the cell cycle. Understanding of RA signaling has increased dramatically over the past decades, but the connection between whole body RA homeostasis and gene regulation in individual cells is still unclear. It has been proposed that cytochrome P450 family 26 (CYP26) enzymes have a role in determining the cellular exposure to RA by inactivating RA in cells that do not need RA. The CYP26 enzymes have been shown to metabolize RA efficiently and they are also inducible by RA in selected systems. However, their expression patterns in different cell types and a mechanistic understanding of their function is still lacking. Based on preliminary kinetic data and protein expression levels, one may predict that if CYP26A1 is expressed in the liver at even very low levels, it will be the major RA hydroxylase in this tissue. As such, it is an attractive pharmacological target for drug development when one aims to increase circulating or cellular RA concentrations. To further the understanding of how CYP26 enzymes contribute to the regulation of RA homeostasis, structural information of the CYP26's, commercially available recombinant enzymes and good specific and sensitive antibodies are needed.

All-trans-retinoic acid (atRA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of atRA via oxidative metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are atRA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from atRA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from atRA. Quantitatively, CYP26B1 had a lower Km (19nM) and Vmax (0.8pmol/min/pmol) than CYP26A1 (Km=50nM and Vmax=10pmol/min/pmol) for formation of 4-OH-RA. The major atRA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2–10 fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of atRA in tissues that function as a barrier for atRA exposure.

Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor been characterized kinetically. We expressed the rCYP26A1 in baculovirus infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion K m 9.4 ± 3.3 nM and V max 11.3 ± 4.3 pmoles/min/pmole P450) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.

All-trans retinoic acid (RA) is a critical signaling molecule and its concentration is tightly regulated. Several P450 enzymes including CYP26A1, CYP2C8, and CYP3A4 have been proposed to be responsible for RA clearance in the liver but their quantitative importance has not been demonstrated. To determine the contribution of CYP26A1 to hepatic clearance of RA, CYP26A1 protein was quantified in 37 human liver microsomes (HLMs). CYP26A1 expression ranged from not detectable to 2.80 pmol/mg microsomal protein. RA clearance by P450 enzymes abundant in human liver was measured in Supersomes®. CYP2C8, CYP3A4, CYP3A5 and CYP3A7 metabolized RA with unbound Km values of 3.4–7.2 μM and Vmax values of 2.3–4.9 pmol/min/pmol P450, but were less efficient than CYP26A1 in clearing RA. Simulations performed for livers with varying P450 expression levels over a range of RA concentrations demonstrated that at both endogenous and therapeutic concentrations of RA, CYP26A1 is the primary enzyme responsible for 4-OHRA formation clearance. HLM incubation data showed that 4-OH RA formation velocity varied from 0.2 to 15.3 pmol/min/mg microsomal protein and velocity in HLMs was significantly correlated (p < 0.01) to CYP26A1, CYP3A4, and CYP3A5 protein content, but not to CYP2C8. When experimental data were scaled to in vivo clearances, the predicted hepatic clearance of RA(0.07 L/min using combined Supersome® data) was similar to the published in vivo clearance of RA. These findings suggest that CYP26A1 is the P450 isoform that should be targeted when designing RA metabolism blocking agents.

All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. atRA is also used as a drug, and synthetic atRA analogs and inhibitors of retinoic acid (RA) metabolism have been developed. The hepatic clearance of atRA is mediated primarily by CYP26A1, but design of CYP26A1 inhibitors is hindered by lack of information on CYP26A1 structure and structure-activity relationships of its ligands. The aim of this study was to identify the primary metabolites of atRA formed by CYP26A1 and to characterize the ligand selectivity and ligand interactions of CYP26A1. On the basis of high-resolution tandem mass spectrometry data, four metabolites formed from atRA by CYP26A1 were identified as 4-OH-RA, 4-oxo-RA, 16-OH-RA and 18-OH-RA. 9-cis-RA and 13-cis-RA were also substrates of CYP26A1. Forty-two compounds with diverse structural properties were tested for CYP26A1 inhibition using 9-cis-RA as a probe, and IC 50 values for 10 inhibitors were determined. The imidazole-and triazole-containing inhibitors [S- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and the peroxisome proliferator-activated receptor ligands rosiglitazone and pioglitazone inhibited CYP26A1 with IC 50 values of 3.7, 4.2, and 8.6 M, respectively. These data demonstrate that CYP26A1 has high ligand selectivity but accepts structurally related nuclear receptor agonists as inhibitors.

Blood specimens from 146 suspected gamma-hydroxybutyrate (GHB) overdose cases, presenting to an emergency department in Washington State over a 12-month period, were analyzed for GHB and other drugs. Of these 146 patients, GHB was confirmed in approximately one-third of the patients (N = 54), sometimes in potentially toxic concentrations. These patients were aged between 17 and 59 years (median 28 years), and 83% were male. Blood GHB concentrations ranged from 29 to 490 mg/L (mean 137 mg/L; median 103 mg/L). In 36 (67%) of the 54 patients, other drugs were additionally detected. Ethanol was measured in 22 (41%) patients, with concentrations ranging from 0.01 to 0.26 g/100 mL (median 0.04 g/100 mL). Other commonly co-administered drugs included 3,4-methylenedioxymethamphetamine, marijuana, methamphetamine, cocaine, and citalopram. Frequently observed clinical symptoms on admission for the GHB overdose group included copious vomiting, ataxia, lack of gag reflex, respiratory depression, mild acute respiratory acidosis, unconsciousness, and sudden altered states of consciousness. Many patients required intubation, and several became combative and required restraints. The majority of patients were discharged within 6 h of hospital admission. However, despite presenting with similar clinical symptoms on admission, GHB was not confirmed in 92 of the 146 overdose patients, suggesting that GHB overdose cases may frequently be indistinguishable from other drug overdoses or medical conditions.

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a65-74, Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gln (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.