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The major quercetin metabolite, quercetin-3-glucuronide, exerts various biological activities, including anti-inflammatory effects. This study aimed to evaluate the metabolic profiles and biological properties of the positional isomers of quercetin monoglucuronides (Q3G, Q7G, Q3’G, and Q4’G) in activated macrophages. In addition to quercetin aglycone, Q7G was more cytotoxic than the other quercetin monoglucuronides (QGs), which corresponded to its lower stability under neutral pH conditions. Q3G was most effective in inhibiting both LPS-dependent induction of IL-6 and RANKL-dependent activation of tartrate-resistant acid phosphatase; however, Q3’G and Q4’G may also help exert biological activities without potential cytotoxicity. The deconjugation efficacy to generate quercetin aglycone differed among QGs, with the highest efficacy in Q3G. These results suggest that the chemical or biological properties and metabolic profiles may depend on the stability of QGs to generate quercetin aglycone using β-glucuronidase.

Vitamin D3 (1) is metabolized by various cytochrome P450 (CYP) enzymes, resulting in the formation of diverse metabolites. Among them, 4α,25-dihydroxyvitamin D3 (6a) and 4β,25-dihydroxyvitamin D3 (6b) are both produced from 25-hydroxyvitamin D3 (2) by CYP3A4. However, 6b is detectable in serum, whereas 6a is not. We hypothesized that the reason for this is a difference in the susceptibility of 6a and 6b to CYP24A1-mediated metabolism. Here, we synthesized 6a and 6b, and confirmed that 6b has greater metabolic stability than 6a. We also identified 4α,24R,25- and 4β,24R,25-trihydroxyvitamin D3 (16a and 16b) as metabolites of 6a and 6b, respectively, by HPLC comparison with synthesized authentic samples. Docking studies suggest that the β-hydroxy group at C4 contributes to the greater metabolic stability of 6b by blocking a crucial hydrogen-bonding interaction between the C25 hydroxy group and Leu325 of CYP24A1.

CYP105A1 from Streptomyces griseolus converts vitamin D3 to its biologically active form, 1α,25-dihydroxy vitamin D3. R73A/R84A mutation enhanced the 1α- and 25-hydroxylation activity for vitamin D3, while M239A mutation generated the 1α-hydroxylation activity for vitamin D2. In this study, the stability of six CYP105A1 enzymes, including 5 variants (R73A/R84A, M239A, R73A/R84A/M239A (=TriA), TriA/E90A, and TriA/E90D), was examined. Circular dichroism analysis revealed that M239A markedly reduces the enzyme stability. Protein fluorescence analysis disclosed that these mutations, especially M239A, induce large changes in the local conformation around Trp residues. Strong stabilizing effect of glycerol was observed. Nondenaturing PAGE analysis showed that CYP105A1 enzymes are prone to self-association. Fluorescence analysis using a hydrophobic probe 8-anilino-1-naphthalenesulfonic acid suggested that M239A mutation enhances self-association and that E90A and E90D mutations, in cooperation with M239A, accelerate self-association with little effect on the stability.

1α,25-Dihydroxyvitamin D3 (abbreviated here as 1,25D3 ) is a hormonally active form of vitamin D3 (D3 ), and is produced from D3 by CYP27 A1-mediated hydroxylation at C25, followed by CYP27B1-mediated hydroxylation at C1. Further hydroxylation of 25D3 and 1,25D3 occurs at C23, C24 and C26 to generate corresponding metabolites, except for 1,25R,26D3 . Since the capability of CYP27B1 to hydroxylate C1 of side-chain-hydroxylated metabolites other than 23S,25D3 and 24R,25D3 has not been examined, we have here explored the role of CYP27B1 in the C1 hydroxylation of a series of side-chain-hydroxylated D3 derivatives. We found that CYP27B1 hydroxylates the R diastereomers of 24,25D3 and 25,26D3 more effectively than the S diastereomers, but shows almost no activity towards either diastereomer of 23,25D3 . This is the first report to show that CYP27B1 metabolizes 25,26D3 to the corresponding 1α-hydroxylated derivative, 1,25,26D3 . It will be interesting to examine the physiological relevance of this finding.

Previously, we reported a FLucN-LXXLL+LBD-FLucC system that detects VDR ligands using split firefly luciferase techniques, ligand binding domain (LBD) of VDR, and LXXLL sequences that interact with LBD after VDR ligand binding. In vivo, 25-hydroxyvitamin D

Active vitamin D form 1a,25-dihydroxtvitamin D3 (1,25(OH)2D3) plays pivotal roles in calcium homeostasis and osteogenesis via its transcription regulation effect via binding to vitamin D receptor (VDR). Mutated VDR often causes hereditary vitamin D-dependent rickets (VDDR) type II, and patients with VDDR-II are hardly responsive to physiological doses of 1,25(OH)2D3. Current therapeutic approaches, including high doses of oral calcium and supraphysiologic doses of 1,25(OH)2D3, have limited success and fail to improve the quality of life of affected patients. Thus, various vitamin D analogues have been developed as therapeutic options. In our previous study, we generated genetically modified rats with mutated Vdr(R270L), an ortholog of human VDR(R274L) isolated from the patients with VDDR-II. The significant reduced affinity toward 1,25(OH)2D3 of rat Vdr (R270L) enabled us to evaluate biological activities of exogenous VDR ligand without 1a-hydroxy group such as 25(OH)D3. In this study, 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exerted much higher affinity for Vdr(R270L) in in vitro ligand binding assay than both 25(OH)D3 and 1,25(OH)2D3. A robust osteogenic activity of AH-1 was observed in Vdr(R270L) rats. Only a 40-fold lower dose of AH-1 than that of 25(OH)D3 was effective in ameliorating rickets symptoms in Vdr(R270L) rats. Therefore, AH-1 may be promising for the therapy of VDDR-II with VDR(R274L).

Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.

Flavin containing monooxygenases (FMOs) represent one of the predominant types of phase I drug metabolizing enzymes (DMEs), and thus play an important role in the metabolism of xeno- and endobiotics for the generation of their corresponding oxides. These oxides often display biological activities, however they are difficult to study since their chemical or biological synthesis is generally challenging even though only small amounts are required to evaluate their efficacy and safety. Previously, we constructed a DME expression system for cytochrome P450, UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) using yeast cells, and successfully produced xenobiotic metabolites in a whole-cell dependent manner. In this study, we developed a heterologous expression system for human FMOs, including FMO1–FMO5, in Saccharomyces cerevisiae and examined its N- and S-oxide productivity. The recombinant yeast cells expressed each of the FMO successfully, and the FMO4 transformant produced N- and S-oxide metabolites at several milligrams per liter within 24 h. This whole-cell dependent biosynthesis enabled the production of N- and S-oxides without the use of the expensive cofactor NADPH. Such novel yeast expression system could be a powerful tool for the production of oxide metabolites.

In the field of drug development, technology for producing human metabolites at a low cost is required. In this study, we explored the possibility of using prokaryotic water-soluble cytochrome P450 (CYP) to produce human metabolites. Streptomyces griseolus CYP105A1 metabolizes various non-steroidal anti-inflammatory drugs (NSAIDs), including diclofenac, mefenamic acid, flufenamic acid, tolfenamic acid, meclofenamic acid, and ibuprofen. CYP105A1 showed 4'-hydroxylation activity towards diclofenac, mefenamic acid, flufenamic acid, tolfenamic acid, and meclofenamic acid. It should be noted that this reaction specificity was similar to that of human CYP2C9. In the case of mefenamic acid, another metabolite, 3'-hydroxymethyl mefenamic acid, was detected as a major metabolite. Substitution of Arg at position 73 with Ala in CYP105A1 dramatically reduced the hydroxylation activity toward diclofenac, flufenamic acid, and ibuprofen, indicating that Arg73 is essential for the hydroxylation of these substrates. In contrast, substitution of Arg84 with Ala remarkably increased the hydroxylation activity towards diclofenac, mefenamic acid, and flufenamic acid. Recombinant Rhodococcus erythrocyte cells expressing the CYP105A1 variant R84A/M239A showed complete conversion of diclofenac into 4'-hydroxydiclofenac. These results suggest the usefulness of recombinant R. erythropolis cells expressing actinomycete CYP, such as CYP105A1, for the production of human drug metabolites.

We have developed an in vitro system to easily examine the affinity for vitamin D receptor (VDR) and CYP24A1-mediated metabolism as two methods of assessing vitamin D derivatives. Vitamin D derivatives with high VDR affinity and resistance to CYP24A1-mediated metabolism could be good therapeutic agents. This system can effectively select vitamin D derivatives with these useful properties. We have also developed an in vivo system including a Cyp27b1-gene-deficient rat (a type I rickets model), a Vdr-gene-deficient rat (a type II rickets model), and a rat with a mutant Vdr (R270L) (another type II rickets model) using a genome editing method. For Cyp27b1-gene-deficient and Vdr mutant (R270L) rats, amelioration of rickets symptoms can be used as an index of the efficacy of vitamin D derivatives. Vdr-gene-deficient rats can be used to assess the activities of vitamin D derivatives specialized for actions not mediated by VDR. One of our original vitamin D derivatives, which displays high affinity VDR binding and resistance to CYP24A1-dependent metabolism, has shown good therapeutic effects in Vdr (R270L) rats, although further analysis is needed.

1α,25-Dihydroxyvitamin D

Two novel 23-fluorinated 25-hydroxyvitamin D3 analogues were synthesized using Inhoffen–Lythgoe diol as a precursor of the CD-ring, efficiently. Introduction of the C23 fluoro group was achieved by the deoxy-fluorination reaction using N,N-diethylaminosulfur trifluoride or 2-pyridinesulfonyl fluoride (PyFluor). Kinetic studies on the CYP24A1-dependent metabolism of these two analogues revealed that (23S)-23-fluoro-25-hydroxyvitamin D3 was more resistant to CYP24A1-dependent metabolism than its 23R isomer.

Acute myeloid leukemia (AML) is one of the deadliest hematological malignancies without effective treatment for most patients. Vitamin D derivatives (VDDs) - active metabolites 1α,25-dihydroxyvitamin D2 (1,25D2) and 1α,25-dihydroxyvitamin D3 (1,25D3) and their analogs - are differentiation-inducing agents which have potential for the therapy of AML. However, calcemic toxicity of VDDs limits their clinical use at doses effective against cancer cells in vivo. Here, we demonstrate that in AML cell cultures, moderate pro-differentiation effects of low concentrations of VDDs can be synergistically enhanced by structurally distinct compounds known to activate the transcription factor Nuclear Factor (Erythroid-derived 2)-Like 2 (NFE2L2 or Nrf2). Particularly, dimethyl fumarate (DMF), which is clinically approved for the treatment of multiple sclerosis and psoriasis, strongly cooperated with 1,25D3, PRI-5100 (19-nor-1,25D2; paricalcitol) and PRI-5202 (a double-point modified 19-nor analog of 1,25D2). The pro-differentiation synergy between VDDs (1,25D3 or PRI-5202) and Nrf2 activators (DMF, tert-butylhydroquinone or carnosic acid) was associated with a cooperative upregulation of the protein levels of the vitamin D receptor (VDR) and Nrf2 as well as increased mRNA expression of their respective target genes. These data support the notion that VDDs and Nrf2 activators synergize in inducing myeloid cell differentiation through the cooperative activation of the VDR and Nrf2/antioxidant response element signaling pathways. We have previously reported that PRI-5202 is more potent by approximately two orders of magnitude than 1,25D3 as a differentiation inducer in AML cell lines. In this study, we found that PRI-5202 was also at least 5-fold less calcemic in healthy mice compared to both its direct precursor PRI-1907 and 1,25D3. In addition, PRI-5202 was remarkably more resistant against degradation by the human 25-hydroxyvitamin D3-24-hydroxylase than both 1,25D2 and 1,25D3. Importantly, using a xenograft mouse model we demonstrated that co-administration of PRI-5202 and DMF resulted in a marked cooperative inhibition of human AML tumor growth without inducing treatment toxicity. Collectively, our findings provide a rationale for clinical testing of low-toxic VDD/DMF combinations as a novel approach for differentiation therapy of AML.

Three 23-hydroxylated vitamin D3 derivatives, which are metabolites of 25-hydroxyvitamin D3 produced by CYP24A1 and a related diastereomer, were efficiently synthesized. Each C23 hydroxy unit was constructed by the Claisen condensation reaction with ethyl acetate or the Grignard reaction with 2-methylallymagnesium chloride. Stereochemistry at the C23 position was determined by a modified Mosher's method. The triene structures were constructed by the Wittig-Horner reaction utilizing the A-ring phosphine oxide moiety.

We have reported that 25-hydroxyvitamin D3 [25(OH)D3] binds to vitamin D receptor and exhibits several biological functions directly in vitro. To evaluate the direct effect of 25(OH)D3 in vivo, we used Cyp27b1 knockout (KO) mice, which had no detectable plasma 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] when fed a diet containing normal Ca and vitamin D. Daily treatment with 25(OH)D3 at 250 μg kg-1 day-1 rescued rachitic phenotypes in the Cyp27b1 KO mice. Bone mineral density, female sexual cycles, and plasma levels of Ca, P, and PTH were all normalized following 25(OH)D3 administration. An elevated Cyp24a1 mRNA expression was observed in the kidneys, and plasma concentrations of Cyp24a1-dependent metabolites of 25(OH)D3 were increased. To our surprise, 1,25(OH)2D3 was detected at a normal level in the plasma of Cyp27b1 KO mice. The F1 to F4 generations of Cyp27b1 KO mice fed 25(OH)D3 showed normal growth, normal plasma levels of Ca, P, and parathyroid hormone, and normal bone mineral density. The curative effect of 25(OH)D3 was considered to depend on the de novo synthesis of 1,25(OH)2D3 in the Cyp27b1 KO mice. This suggests that another enzyme than Cyp27b1 is present for the 1,25(OH)2D3 synthesis. Interestingly, the liver mitochondrial fraction prepared from Cyp27b1 KO mice converted 25(OH)D3 to 1,25(OH)2D3. The most probable candidate is Cyp27a1. Our findings suggest that 25(OH)D3 may be useful for the treatment and prevention of osteoporosis for patients with chronic kidney disease.

Cytochrome P450 (CYP) monooxygenases play critical roles in determining the toxicity of polychlorinated biphenyls (PCBs) in mammals. Hydroxylation of PCBs by these enzymes leads to increased water solubility, promoting the elimination of PCBs from the body. The CYP1 family is mainly responsible for metabolizing PCBs that exhibit a dioxin-like toxicity. Although the dioxin-like PCB 3,3',4,4'-tetrachlorobiphenyl (CB77) is abundant in the environment and accumulates in organisms, information on CB77 metabolism by CYP1A1s is limited. In this study, recombinant rat CYP1A1 metabolized CB77 to 4'-hydroxy (OH)-3,3',4,5'-tetrachlorobiphenyl (CB79) and 4'-OH-3,3',4-trichlorobiphenyl (CB35), whereas human CYP1A1 produced only 4'-OH-CB79. Rat CYP1A1 exhibited much higher metabolizing activity than human CYP1A1 because CB77 was stably accommodated in the substrate-binding cavity of rat CYP1A1 and was close to its heme. In a rat CYP1A1 mutant with two human-type amino acids, the production of 4'-OH-CB79 decreased, whereas that of the dechlorinated metabolite 4'-OH-CB35 increased. These results are explained by a shift in the CB77 positions toward the heme. This study provides insight into the development of enzymes with high metabolizing activity and clarifies the structural basis of PCB metabolism, as dechlorination contributes to a drastic decrease in dioxin-like toxicity.

The microsomal cytochrome P450 3A4 (CYP3A4) and mitochondrial cytochrome P450 24A1 (CYP24A1) hydroxylating enzymes both metabolize vitamin D and its analogs. The three-dimensional (3D) structure of the full-length native human CYP3A4 has been solved, but the respective structure of the main vitamin D hydroxylating CYP24A1 enzyme is unknown. The structures of recombinant CYP24A1 enzymes have been solved; however, from studies of the vitamin D receptor, the use of a truncated protein for docking studies of ligands led to incorrect results. As the structure of the native CYP3A4 protein is known, we performed rigid docking supported by molecular dynamic simulation using CYP3A4 to predict the metabolic conversion of analogs of 1,25-dihydroxyvitamin D2 (1,25D2). This is highly important to the design of novel vitamin D-based drug candidates of reasonable metabolic stability as CYP3A4 metabolizes ca. 50% of the drug substances. The use of the 3D structure data of human CYP3A4 has allowed us to explain the substantial differences in the metabolic conversion of the side-chain geometric analogs of 1,25D2. The calculated free enthalpy of the binding of an analog of 1,25D2 to CYP3A4 agreed with the experimentally observed conversion of the analog by CYP24A1. The metabolic conversion of an analog of 1,25D2 to the main vitamin D hydroxylating enzyme CYP24A1, of unknown 3D structure, can be explained by the binding strength of the analog to the known 3D structure of the CYP3A4 enzyme.

CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher Tmax value than others. When 23S,25(OH)2D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)3D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)3D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)3D3 via 23,25(OH)2D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)3D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)3D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 μg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.

We previously revealed that the anti-proliferative activity of 25-hydroxyvitamin D3 (25(OH)D3) in human prostate PZ-HPV-7 cells depends on the direct action of 25(OH)D3 through the vitamin D receptor (VDR). We then attempted to confirm the direct action of 25(OH)D3 in vivo using Cyp27b1 knockout (KO) mice. Daily administration of 25(OH)D3 at 250 μg/kg bw/day rescued the rachitic conditions in Cyp27b1 KO mice. The plasma levels of calcium, phosphorus, and parathyroid hormonea as well as the bone mineral density and female sexual cycle were normalized via the administration of 25(OH)D3. These results strongly suggest the direct action of 25(OH)D3. However, to our surprise, normal levels of 1a,25(OH)2D3 were detected in the plasma of Cyp27b1 KO mice, probably due to Cyp27a1, which has a weak 1a-hydroxylation activity toward 25(OH)D3. Next, we generated a novel in vivo system using genetically modified (GM) rats deficient in the Cyp27b1 or Vdr gene to reveal the molecular mechanisms of vitamin D action. Human type II rickets model rats with mutant Vdr (R270L), which recognizes 1,25(OH)2D3 with an affinity equivalent to that of 25(OH)D3, were also generated. Cyp27b1-knockout (KO), Vdr-KO, and Vdr (R270L) rats showed symptoms of rickets, including growth retardation and abnormal bone formation. Among these model animals, Cyp27b1-KO rats had notably low levels of calcium in the blood and the most severe growth retardation, while Vdr-KO rats showed abnormal skin formation and alopecia. Administration of 25(OH)D3 restored rickets symptoms in Cyp27b1-KO and Vdr (R270L) rats. As shown in Cyp27b1-KO mice, 1,25(OH)2D3 was also synthesized in Cyp27b1-KO rats. In contrast, the effects of 25(OH)D3 on Vdr (R270L) rats strongly suggest that 25(OH)D3 exerts a direct action via VDR-genomic pathways. These results suggest that our novel in vivo system containing three different types of GM rats is useful for elucidating the molecular mechanism of vitamin D action.

Sesamin is a major lignan in sesame, and its biological effects such as antioxidant effect, anti-carcinogenic effects, and suppression of hypertension have been extensively studied by many researchers. However, its metabolic pathways and metabolic enzymes in human bodies have not been identified. Recently we demonstrated that CYP2C9 was the most important cytochrome P450 isoform in human liver. Next, we focused on metabolism of sesamin mono-catechol by cytochrome P450 or UDP-glucuronosyltransferase (UGT). Further catecholization of sesamin mono-catechol by cytochrome P450 enhances its anti-oxidant activity, whereas glucuronidation by UGT strongly reduces anti-oxidant activity. In human liver microsomes, glucuronidation activity toward sesamin mono-catechol was much higher than the di-catecholization activity. In contrast, both activities were similar in rat liver microsomes. These results suggest a large species-based difference between humans and rats in sesamin metabolism. In vitro studies using 10 individual human liver microsomes suggested that UGT2B7 was responsible for glucuronidation of sesamin mono-catechol in human liver. In addition, we observed a significant methylation activity toward sesamin mono-catechol by catechol O-methyl transferase (COMT) in human liver cytosol. Based on these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in human liver.

A recent report demonstrated that sesamin strongly and non-competitively inhibits S-warfarin 7-hydroxylation activity in human liver microsomes with a Ki value of 0.2 μM. This finding suggests that sesamin predominantly binds to CYP2C9 at another site for which it has a higher affinity than its affinity for the active site, thereby inhibiting the activity of CYP2C9 non-competitively. In this study, we found that sesamin competitively inhibited the 7-hydroxylation activity of S-warfarin in human liver microsomes with a Ki value of 15.7 μM. In addition, the recombinant CYP2C9-dependent 7-hydroxylation activity of S-warfarin was competitively inhibited by sesamin with a Ki value of 13.1 μM. These results are consistent with the fact that sesamin is a good substrate of CYP2C9, and its activity follows Michaelis-Menten kinetics. As the plasma concentration of sesamin after its administration is usually lower than 0.01 μM, the inhibition of S-warfarin metabolism by sesamin does not appear to be severe.

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 25-hydroxyvitamin D3 (25(OH)D3) and ligand-independent effects of the VDR. Here, we describe a novel in vivo system using genetically modified rats deficient in the Cyp27b1 or Vdr genes. Type II rickets model rats with a mutant Vdr (R270L), which recognizes 1,25(OH)2D3 with an affinity equivalent to that for 25(OH)D3, were also generated. Although Cyp27b1-knockout (KO), Vdr-KO, and Vdr (R270L) rats each showed rickets symptoms, including abnormal bone formation, they were significantly different from each other. Administration of 25(OH)D3 reversed rickets symptoms in Cyp27b1-KO and Vdr (R270L) rats. Interestingly, 1,25(OH)2D3 was synthesized in Cyp27b1-KO rats, probably by Cyp27a1. In contrast, the effects of 25(OH)D3 on Vdr (R270L) rats strongly suggested a direct action of 25(OH)D3 via VDR-genomic pathways. These results convincingly suggest the usefulness of our in vivo system.

Experimental data indicate that low-calcemic vitamin D derivatives (VDDs) exhibit anticancer properties, both in vitro and in vivo. In our search for a vitamin D analog as potential anticancer agent, we investigated the influence of chirality in the side chain of the derivatives of 1,25-dihydroxyergocalciferol (1,25D2) on their activities. In this study, we synthesized modified analogs at the side chain and the A-ring, which differed from one another in their absolute configuration at C-24, namely (24S)- and (24R)-1,25-dihydroxy-19-nor-20a-homo-ergocalciferols (PRI-5105 and PRI-5106, respectively), and evaluated their activity. Unexpectedly, despite introducing double-point modifications, both analogs served as very good substrates for the vitamin D-hydroxylating enzyme. Irrespective of their absolute C-24 configuration, PRI-5105 and PRI-5106 showed relatively low resistance to CYP24A1-dependent metabolic deactivation. Additionally, both VDDs revealed a similar antiproliferative activity against HT-29 colorectal cancer cells which was higher than that of 1,25D3, the major biologically active metabolite of vitamin D. Furthermore, PRI-5105 and PRI-5106 significantly enhanced the cell growth-inhibitory activity of 5-fluorouracil on HT-29 cell line. In conclusion, although the two derivatives showed a relatively high anticancer potential, they exhibited undesired high metabolic conversion.

Recently, we successfully generated a novel detection system for vitamin D receptor (VDR) ligands in vivo and in vitro, using a split-luciferase technique called the LucN-LBD-LucC biosensor that is a chimeric fusion protein of firefly luciferase with the ligand binding domain (LBD) of VDR. In this system, the luciferase light intensity of the LucN-LBD-LucC biosensor was decreased by binding of VDR ligands. Although this system is quite useful for evaluation of VDR ligands in a short time, the sensitivity of the LucN-LBD-LucC biosensor is not high enough. In this study, LXXLL motif peptides involved in the interaction between LBD and coactivators, such as the steroid receptor coactivator-1 (SRC-1), transcriptional intermediary factor 2 (TIF2), and the vitamin D receptor interacting protein 205 (DRIP205) were each inserted between LucN and LBD of the LucN-LBD-LucC biosensor. Surprisingly, the resulting LucN-LXXLL-LBD-LucC biosensor increased the light intensity in response to natural VDR ligands. This high-sensitivity biosensor system may be a powerful tool for discovery of high-affinity ligands for the mutant VDR. In addition, we have successfully estimated the activity of the wild-type and mutant CYP27B1 using the LucN-LXXLL-LBD-LucC biosensor in living cells within 90 min.

Our previous study revealed that the 2α-substituted vitamin D analog 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exhibited a higher osteocalcin promoter transactivation activity in human osteosarcoma cells and a greater effect on bone mineral density in a rat model of osteoporosis than 1α,25(OH)2D3 without increasing blood calcium concentration. Thus, we hypothesized that AH-1 could be a promising therapeutic agent for osteoporosis without any serious side effects. In this study, we compared the CYP24A1-dependent metabolism of AH-1 with that of 1α,25(OH)2D3. The resistance to CYP24A1-dependent metabolism could be an important property of vitamin D analogs in prolonging their biological effects. A kinetic analysis was performed using a membrane fraction prepared from recombinant E. coli expressing human CYP24A1. The kcat/Km (μM-1 min-1) value for AH-1 was 31% of that for 1α,25(OH)2D3, suggesting that AH-1 is not as resistant to CYP24A1-dependent metabolism as the other C2-substituted vitamin D analogs such as eldecalcitol [2β-hydroxypropoxy-1α,25(OH)2D3]. The major metabolite of AH-1 was the 24R-hydroxylated metabolite, which had high vitamin D receptor (VDR) binding affinity and high HL-60 cell differentiation activity similar to AH-1 itself. In contrast, 1α,25(OH)2D3 was metabolized by multistep monooxygenation reactions, which led to the loss of affinity for VDR. Thus, the greater therapeutic effects of AH-1 than those of 1α,25(OH)2D3 in in vivo studies using osteoporosis rat models may be due to 24R-hydroxy-AH-1 whose VDR affinity was 91% of that of AH-1.

Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Androgen is beneficial for the prostate with normal functions but creates a risk for prostate cancer progression. How androgen receptor (AR) mediates these various actions of androgen remains elusive. AR conserves a phosphorylation motif within its ligand-binding domain throughout species. Here, we have found AR phosphorylated at Ser815 (P-AR) is expressed in normal tissues of both human and mouse prostates. P-AR begins being expressed in association with prostatic development and decreases its expression levels by castration in the mouse prostate. Functional analysis of AR in the prostate cancer PC-3 cells showed ligand-induced AR nuclear translocation and transactivation were disturbed by its phosphorylation at Ser815. Moreover, P-AR suppressed oncogenic AKT signaling suggesting AR has a suppression function for prostate cancer development. In fact, AR phosphorylation levels progressively decrease in human prostates as cancer worsens. These findings showed androgen might utilize P-AR to self-antagonize oncogenic signals and cancer progression believed to be regulated by non-phosphorylated AR (NonP-AR). By differing its target genes and signal regulations from those of NonP-AR, P-AR co-expression with NonP-AR may be the molecular basis for androgen to balance its actions and to control disease developments.

Two 24-fluoro-25-hydroxyvitamin D3 analogues (3,4) were synthesized in a convergent manner. The introduction of a stereocenter to the vitamin D3 side-chain C24 position was achieved via Sharpless dihydroxylation, and a deoxyfluorination reaction was utilized for the fluorination step. Comparison between (24R)- and (24S)-24-fluoro-25-hydroxyvitamin D3 revealed that the C24-R-configuration isomer 4 was more resistant to CYP24A1-dependent metabolism than its 24S-isomer 3. The new synthetic route of the CYP24A1 main metabolite (24R)-24,25-dihydroxyvitamin D3 (6) and its 24S-isomer (5) was also studied using synthetic intermediates (30,31) in parallel.

Recently, in addition to carboxylesterases (CESs), we found that arylacetamide deacetylase (AADAC) plays an important role in the metabolism of some clinical drugs. In this study, we screened for food-related natural compounds that could specifically inhibit human AADAC, CES1, or CES2. AADAC, CES1, and CES2 activities in human liver microsomes were measured using phenacetin, fenofibrate, and procaine as specific substrates, respectively. In total, 43 natural compounds were screened for their inhibitory effects on each of these enzymes. Curcumin and quercetin showed strong inhibitory effects against all three enzymes, whereas epicatechin, epicatechin gallate (ECg), and epigallocatechin gallate (EGCg) specifically inhibited AADAC. In particular, ECg and EGCg showed strong inhibitory effects on AADAC (IC50 values: 3.0 ± 0.5 and 2.2 ± 0.2 μM, respectively). ECg and EGCg also strongly inhibited AADAC-mediated rifampicin hydrolase activity in human liver microsomes with IC50 values of 2.2 ± 1.4 and 1.7 ± 0.4 μM, respectively, whereas it weakly inhibited p-nitrophenyl acetate hydrolase activity, which is catalyzed by AADAC, CES1, and CES2. Our results indicate that ECg and EGCg are potent inhibitors of AADAC.

Previously, we found that 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 (AH-1) showed higher osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells and a greater therapeutic effect on ovariectomized (OVX) rats for enhancing bone mineral density than those of 1α,25(OH)2D3 without hypercalcemic side effects in vivo. Although CYP24A1 catalyzes multi-step oxidations toward the CD-ring side chain of the active vitamin D3 [1α,25(OH)2D3], the CYP24A1-dependent metabolism of AH-1 tended to stop at the first step hydroxylation at the C24-position of AH-1. Interestingly, the metabolite 24-hydroxy-AH-1 [24(OH)AH-1] showed potent VDR binding affinity, and the new chiral center of the 24-position might be the 24R configuration compared with the process of the natural 1α,25(OH)2D3 catabolism. This time, (24R)-2α-[2-(tetrazol-2-yl)ethyl]-1α,24,25-trihydroxyvitamin D3 [(24R-OH)AH-1] was synthesized as a candidate for the major metabolite of AH-1 using the Trost Pd-mediated coupling reaction between A-ring and CD-ring precursors to identify the metabolite and evaluate its biological activity. We confirmed that the CYP24A1-dependent major metabolite of AH-1 was (24R-OH)AH-1 by HPLC analyses.

Background:S-equol, which is enantioselectively produced from daidzein by gut microbiota, has been suggested as a chemopreventive agent against type 2 diabetes mellitus (T2DM), but the underlying mechanisms remain unclear.Objective: We investigated the effects of S-equol on pancreatic β-cell function.Methods: β-Cell growth and insulin secretion were evaluated with male Institute of Cancer Research mice and isolated pancreatic islets from the mice, respectively. The mechanisms by which S-equol stimulated β-cell response were examined in INS-1 β-cells. The effect of S-equol treatment on β-cell function was assessed in low-dose streptozotocin-treated mice. S-equol was used at 10 μmol/L for in vitro and ex vivo studies and was administered by oral gavage (20 mg/kg, 2 times/d throughout the experimental period) for in vivo studies.Results:S-equol administration for 7 d increased Ki67-positive β-cells by 27% (P < 0.01) in mice. S-equol enantioselectively enhanced glucose-stimulated insulin secretion in mouse pancreatic islets by 41% (P < 0.001). In INS-1 cells, S-equol exerted stronger effects than daidzein on cell growth, insulin secretion, and cAMP-response element (CRE)-mediated transcription. These S-equol effects were diminished by inhibiting protein kinase A. The effective concentration of S-equol for stimulating cAMP production at the plasma membrane was lower than that for phosphodiesterase inhibition. S-equol-stimulated CRE activation was negatively controlled by the knockdown of G-protein α subunit group S (stimulatory) and positively controlled by that of G-protein-coupled receptor kinase-3 and -6. Compared with vehicle-treated controls, S-equol gavage treatment resulted in an increase in β-cell mass of 104% (P < 0.05), a trend toward high plasma insulin concentrations (by 118%; P = 0.06), and resistance to hyperglycemia after streptozotocin treatment (78% of AUC after glucose challenge; P < 0.01). S-equol administration significantly increased the number of Ki67-positive proliferating β-cells by 62% (P < 0.01) and decreased that of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic β-cells by 75% (P < 0.05).Conclusions: Our results show that S-equol boosts β-cell function and prevents hypoglycemia in mice, suggesting its potential for T2DM prevention.

Our previous studies revealed that CYP105A1 can convert vitamin D3 (VD3) to its active form, 1α,25-dihydroxyvitamin D3 (1,25D3). Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity; the activity of double variants R73A/R84A and R73A/R84V was more than 100-fold higher than that of the wild type of CYP105A1. In contrast, these variants had a low ability to convert vitamin D2 (VD2) to 1α,25-dihydroxyvitamin D2 (1,25D2), whereas they catalyzed the sequential hydroxylation at positions C25 and C26 to produce 25,26D2. A comparison of the docking models of 25D2 and 25D3 into the substrate-binding pocket of R73A/R84A suggests that the side chain of the Met239 inhibits the binding of 25D2 for 1α-hydroxylation. Therefore, the Met239 residue of R73A/R84A was substituted for Ala. As expected, the triple variant R73A/R84A/M239A showed a 22-fold higher 1α-hydroxylation activity towards 25D2. To the best of our knowledge, this is the first report on the generation of microbial cytochrome P450 that converts VD2 to 1,25D2 via 25D2.

Hereditary vitamin D-resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR) gene. Arg274 located in the ligand binding domain (LBD) of VDR is responsible for anchoring 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) by forming a hydrogen bond with the 1α-hydroxyl group of 1α,25(OH)2D3. The Arg274Leu (R274L) mutation identified in patients with HVDRR causes a 1000-fold decrease in the affinity for 1α,25(OH)2D3, and dramatically reduces vitamin D- related gene expression. Recently, we successfully constructed fusion proteins consisting of split-luciferase and LBD of the VDR. The chimeric protein LucC-LBD-LucN, which displays the C-terminal domain of luciferase (LucC) at its N-terminus, can detect and discriminate between VDR agonists and antagonists. The LucC-LBD (R274L)-LucN was constructed to screen high-affinity ligands for the mutant VDR (R274L). Of the 33 vitamin D analogs, 5 showed much higher affinities for the mutant VDR (R274L) than 1α,25(OH)2D3, and 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-(OH)2D3 showed the highest affinity. These compounds might be potential therapeutics for HVDRR caused by the mutant VDR (R274L).

The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25D3), plays an important role in the maintenance of calcium (Ca) homeostasis, bone formation, and cell proliferation and differentiation via nuclear vitamin D receptor (VDR). It is formed by the hydroxylation of vitamin D at the 1α position by 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1) in the kidney. However, Cyp27b1-/- mice, deficient in CYP27B1, and VDR-deficient mice (Vdr-/-) have not been extensively examined, particularly in a comparative framework. To clarify the physiological significance of 1α,25D3 and VDR, we produced Cyp27b1-/- mice and compared their phenotypes with those of Vdr-/- mice. Cyp27b1-/- mice exhibited hypocalcemia, growth defects, and skeletogenesis dysfunction, similar to Vdr-/- mice. However, unlike Cyp27b1-/- mice, Vdr-/- mice developed alopecia. Cyp27b1-/- mice exhibited cartilage mass formation and had difficulty walking on hindlimbs. Furthermore, a phenotypic analysis was performed on Cyp27b1-/- mice provided a high Ca diet to correct for the Ca metabolic abnormality. In addition, the effects of 1α,25D3 that are not mediated by Ca metabolic regulatory activity were investigated. Even when the blood Ca concentration was corrected, abnormalities in growth and cartilage tissue formation did not improve in Cyp27b1-/- mice. These results suggested that 1α,25D3 directly controls chondrocyte proliferation and differentiation. Using Cyp27b1-/- mice produced in this study, we can analyze the physiological effects of novel vitamin D derivatives in the absence of endogenous 1α,25D3. Accordingly, this study provides a useful animal model for the development of novel vitamin D formulations that are effective for the treatment and prevention of osteoporosis.

During our ongoing studies of vitamin D, we focused on the vitamin D

There is no suitable medicine for rickets caused by mutation of vitamin D receptor (VDR). In the present study, we found the promising new compounds for this disease using bioluminescence assay.

Recently, we found that 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 showed higher osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells and a greater therapeutic effect in ovariectomized (OVX) rats in vivo than those of active vitamin D3, 1α,25(OH)2D3. We were interested in introducing a heterocyclic ring to the C2 position of the seco-steroidal structure via an alkyl linker, and four novel C2-(3-tetrazolylpropyl) substituted 1α,25-dihydroxy-19-norvitamin D3 analogs, 2α-[3-(tetrazol-1-yl)propyl]-, 2β-[3-(tetrazol-1-yl)propyl]-, 2α-[3-(tetrazol-2-yl)propyl]-, and 2β-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 were synthesized. Among them, 2α-[3-(tetrazol-1-yl)propyl]-19-nor-1α,25(OH)2D3 showed weak binding affinity for human vitamin D receptor (hVDR) (2.6% of 1α,25(OH)2D3 and ca. 15% of 19-nor-1α,25(OH)2D3) and weak VDR transactivation activity in HOS cells (EC50 7.3nM, when 1α,25(OH)2D30.23nM). Although the other three compounds could not act as VDR binders by evaluation of the competition assays, 2α-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 showed weak transactivation activity (EC50 12.5nM). Metabolic stability of the 2α-substituted compounds 2α-[3-(tetrazol-1-yl)propyl]- and 2α-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 was higher than that of the 2β-substituted counterparts 2β-[3-(tetrazol-1-yl)propyl]- and 2β-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 against human CYP24A1. Introduction of a tetrazole ring to the C2-position of the 19-norvitamin D3 skeleton with the propyl linker led to weak VDR agonistic activity with stability against CYP24A1 metabolism.

An improved convergent strategy was developed for the synthesis of the previously obtained side-chain extended and rigidified analogs of 1α,25-dihydroxyvitamin D2, PRI-1906 and PRI-1907. New (24Z) geometric isomers of the analogs, PRI-1916 and PRI-1917, were also obtained and identified. These side-chain isomers were separable by flash chromatography, as C-25 alcohols, from the synthetic precursors of PRI-1906 and PRI-1907, respectively. The structures of new analogs were determined by advanced techniques of 1H and 13C NMR, including COSY, HSQC and HMBC sequences. Binding affinities of the geometric analogs PRI-1906 and PRI-1916 and their respective C-26, C-27 homologs PRI-1907 and PRI-1917 for the full-length human vitamin D receptor were determined by a fluorescence polarization competition assay. The binding affinity of (24Z) methyl analog PRI-1906 was much higher than that of (24E) analog PRI-1906, while the affinity of (24Z) ethyl analog PRI-1917 was lower than that of the respective PRI-1907. Investigation of the metabolism of these compounds by human CYP24A1 revealed they are much more resistant to CYP24A1 than 1α,25-dihydroxyvitamin D2, indicating they could have longer-term biological effects on target tissues.