Expression of microsomal lanosterol 14alpha-demethylase (CYP51) in an engineered soluble monomeric form

We prepared a soluble monomeric form of bovine cytochrome P450 lanosterol 14α-demethylase (CYP51), which in mammals is a ubiquitously expressed membrane protein of the endoplasmic reticulum. We constructed two variants of bovine CYP51 (bCYP51) with different truncations and modifications in their N-terminal membrane-spanning domains. Both of these were expressed in Escherichia coli at levels of 500 nmol/l. The protein variants were purified and tested for the solubility in the absence of detergent. Variant bCYP51-d1 exhibited ∼10-fold better solubility over variant bCYT51-d2. The bCYP51-d1 eluted as a single peak in size-exclusion chromatography, corresponding to its monomeric form. The activity of bCYP51-d1 is similar to that of recombinant human CYP51 with a non-truncated membrane-spanning region. High solubility and low tendency to non-specific oligomer formation make bCYP51-d1 a promising candidate for successful crystallization, which may finally allow the structural determination of this important mammalian enzyme.     

Design, synthesis, and antifungal activities in vitro of novel tetrahydroisoquinoline compounds based on the structure of lanosterol 14alpha-demethylase (CYP51) of fungi

Novel tetrahydroisoquinoline compounds were designed by coupling structure-based de novo design based on the structure of lanosterol 14alpha-demethylase (CYP51). The chemical synthesis and the antifungal activities in vitro of them were reported. The results exhibited that all of the lead compounds showed potent antifungal activities, in which compounds 6 and 7 had equal or stronger antifungal activities against five test fungi than that of fluconazole. The studies presented here provided the antifungal lead compounds. The affinity of the lead molecules for CYP51 was mainly attributed to their non-bonding interaction with the apoprotein, which was different from the azole antifungal agents.     

Substrate preferences and catalytic parameters determined by structural characteristics of sterol 14alpha-demethylase (CYP51) from Leishmania infantum

Leishmaniasis is a major health problem that affects populations of ～90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/function characterization of sterol 14α-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14α-methyl group from sterol precursors. The reaction is essential for membrane biogenesis and therefore has great potential to become a target for antileishmanial chemotherapy. Although L. infantum CYP51 prefers C4-monomethylated sterol substrates such as C4-norlanosterol and obtusifoliol (V(max) of ～10 and 8 min(-1), respectively), it is also found to 14α-demethylate C4-dimethylated lanosterol (V(max) = 0.9 min(-1)) and C4-desmethylated 14α-methylzymosterol (V(max) = 1.9 min(-1)). Binding parameters with six sterols were tested, with K(d) values ranging from 0.25 to 1.4 μM. Thus, L. infantum CYP51 is the first example of a plant-like sterol 14α-demethylase, where requirements toward the composition of the C4 atom substituents are not strict, indicative of possible branching in the postsqualene portion of sterol biosynthesis in the parasite. Comparative analysis of three CYP51 substrate binding cavities (Trypanosoma brucei, Trypanosoma cruzi, and L. infantum) suggests that substrate preferences of plant- and fungal-like protozoan CYP51s largely depend on the differences in the enzyme active site topology. These minor structural differences are also likely to underlie CYP51 catalytic rates and drug susceptibility and can be used to design potent and specific inhibitors.     

Design,synthesis and evaluation of benzoheterocycle analogues as potent antifungal agents targeting CYP51

To further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compound 1, a series of benzoheterocycle analogues were designed, synthesized and evaluated for their in vitro antifungal activity. The most promising compounds 13s and 14a exhibited excellent antifungal activity against C. albicans, C. neoformans, A. fumigatus and fluconazole-resistant C. albicans strains, that was superior or comparable to those of the reference drugs fluconazole and voriconazole. GC–MS analyses suggested that the novel compound 13s might have a similar mechanism to fluconazole by inhibiting fungal lanosterol 14α-demethylase (CYP51). Furthermore, compounds 13s and 14a exhibited low inhibition profiles for various human cytochrome P450 isoforms as well as excellent blood plasma stability.     

Design,synthesis and biological evaluation of amide-pyridine derivatives as novel dual-target (SE,CYP51) antifungal inhibitors

Based on the analysis of the squalene cyclooxygenase (SE) and 14α-demethylase (CYP51) inhibitors pharmacophore feature and the dual-target active sites, a series of compounds with amide-pyridine scaffolds have been designed and synthesized to treat the increasing incidence of drug-resistant fungal infections. In vitro evaluation showed that these compounds have a certain degree of antifungal activity. The most potent compounds 11a, 11b with MIC values in the range of 0.125–2 μg/ml had a broad-spectrum antifungal activity and exhibited excellent inhibitory activity against drug-resistant pathogenic fungi. Preliminary mechanism studies revealed that the compound 11b might play an antifungal role by inhibiting the activity of SE and CYP51. Notably compounds did not show the genotoxicity through plasmid binding assay. Finally, this study of molecular docking, ADME/T prediction and the construction of 3D QSAR model were performed. These results can point out the direction for further optimization of the lead compound.     

Design,synthesis, and structure-activity relationship studies of novel tetrazole antifungal agents with potent activity,broad antifungal spectrum and high selectivity

In this letter, we report our efforts to design, synthesize and evaluate biological activities of a series of novel hybridized compounds containing 1-tetrazole and 4-pyridinyl-1,2,4-triazole-3-one. An analysis of structure-activity data indicates that the target compounds with bulky and hydrophobic side chains exhibited stronger activities against the Candida spp and Cryptococcus neoformans tested than those of fluconazole and racemic VT-1161. Furthermore, 13k and 13ad were active against Microsporum gypseum, which was resistant to racemic VT-1161. In addition, 13k, 13ac and 13ad, with good in vitro activities against all of pathogenic fungi tested except for Aspergillus fumigatus, had no inhibition of human CYP3A4, suggesting a low risk of drug-drug interactions.     

Dynamics of CYP51: implications for function and inhibitor design

Sterol 14α‐demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane‐bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane‐bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen‐bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo

Aiming at the development of new drugs for the treatment of prostate cancer, the effects of steroidal compounds and one non-steroidal substance on androgen biosynthesis were evaluated in vitro and in vivo. Sa 40 [17-(5-pyrimidyl)androsta-5,16-diene-3beta-ol], its 3-acetyl derivate Sa 41 and BW 19 [3,4-dihydro-2-(4-imidazolylmethyl)-6-methoxy-1-methyl-naphthalene] are compounds from our group, which have been developed as inhibitors of CYP 17 (17alpha-hydroxylase-C17, 20-lyase, the key enzyme in androgen biosynthesis). They have been compared with CB 7598 [abiraterone: 17-(3-pyridyl)androsta-5,16-diene-3beta-ol], its 3-acetyl compound CB 7630 and ketoconazole, compounds which already have been used clinically. The most potent compound toward human CYP 17 (testicular microsomes) was Sa 40 (IC(50) value of 24 nM), followed by Sa 41, CB 7598, BW 19, CB 7630 and ketoconazole. Sa 40 shows a type II difference spectrum and a non-competitive type of inhibition (K(i) value of 16 nM). No recovery of enzyme activity was observed after preincubation of CYP 17 with Sa 40 and subsequent charcoal treatment. In Escherichia coli cells coexpressing human CYP 17 and NADPH-P450 reductase, Sa 40 was more active than CB 7598 and BW 19, whereas the acetyl compounds were not active. The latter three compounds were equally active towards rat CYP 17. Male Sprague-Dawley (SD) rats were administered daily for 14 days BW 19 and the acetyl derivatives Sa 41 and CB 7630 as prodrugs (0.1 mmol/kg intraperitoneally). The test compounds strongly reduced plasma testosterone concentration, as well as prostate and seminal vesicles weights. They showed moderate inhibitory effects on the weights of levator ani, bulbocavernosus and testes, whereas they led to an increase in adrenal and pituitary weights. The only exception was BW 19 which did not change pituitary weights. Based on its superiority on the human enzyme, it was concluded that Sa 40 in its 3beta-acetate form (Sa 41) could be a promising candidate for clinical evaluation.     

Effect of a newly developed ketolide antibiotic, telithromycin, on metabolism of theophylline and expression of cytochrome P450 in rats

The effects of a newly-developed ketolide antibiotic, telithromycin, on the metabolism of theophylline and the expression of hepatic cytochrome P450 (CYP) 1A2 and CYP3A2 were investigated in rats. Telithromycin at a high dose (100 mg/kg of body weight) was injected intraperitoneally once a day for 3 days. Twenty-four hours (day 4) after the final administration of telithromycin, theophylline (10 mg/kg) was administered intravenously. The presence of telithromycin significantly delayed the disappearance of theophylline from plasma. Parameters related to the pharmacokinetic interaction between theophylline and telithromycin were examined by noncompartmental methods. A significant decrease in the systemic clearance of theophylline was observed in the presence of telithromycin. Pretreatment with telithromycin significantly decreased the metabolic clearance of the major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, with no change in the renal clearance of theophylline, suggesting that the decreased systemic clearance of theophylline by telithromycin is due to reduction of their metabolic clearance. Pretreatment with telithromycin significantly decreased the activity of 7-ethoxyresorufin O-deethylation and testosterone 6 beta-hydroxylation, suggesting that telithromycin decreases the activity of hepatic CYP1A2 and CYP3A2. Western blot analysis revealed that telithromycin significantly decreased the protein levels of CYP1A2 and CYP3A2 in the liver, which could explain the observed decreases in the systemic clearance of theophylline and metabolic clearance of 1-methyluric acid and 1,3-dimethyluric acid. The present study suggests that telithromycin at the dose used in this study alters the pharmacokinetics and metabolism of theophylline, due to reductions in the activity and expression of hepatic CYP1A2 and CYP3A2.     

Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5

Two acidic residues, Glu-48 and Glu-49, of cytochrome b₅ (b₅) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl)carbodiimide, we obtained cross-linked heterodimers of b₅ and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b₅ double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b₅ complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: ⁸⁴EVLIKK⁸⁹-b₅: ⁵³EQAGGDATENFEDVGHSTDAR⁷³ and CYP17A1-R347K: ³⁴¹TPTISDKNR³⁴⁹-b₅: ⁴⁰FLEEHPGGEEVLR⁵². Using these two sites of interaction and Glu-48/Glu-49 in b₅ as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b₅ complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b₅, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis.     

C(alpha)-hydroxymethyl methionine: synthesis, optical resolution and crystal structure of its (+)-N(alpha)-benzoyl derivative.

(R, S)-Methionine was transformed into C(alpha)-hydroxymethyl methionine by a route involving C(alpha)-hydroxymethylation of 2-phenyl-4-methylthioethyl-5-oxo-4,5-dihydro-1,3-oxazole. The absolute configuration of (-)-C(alpha)-hydroxymethyl methionine was elucidated to be (S) by chemical correlation with (S) (-)-C(alpha)-ethyl serine. Absolute structure determination (by single crystal X-ray diffraction) on N(alpha)-benzoyl-C(alpha)-hydroxymethyl methionine confirmed the (R)-configuration for the (+)-enantiomer. In addition, the X-ray diffraction analysis showed that the C(alpha,alpha)-disubstituted glycyl residue adopts the fully extended (C5) conformation.     

Higher order structure of (1,3)-beta-D-glucans and its influence on their biological activities and complexation abilities

(1,3)-beta-D-Glucans form a group of biologically active biopolymers that exist in different structural organizations depending on the environmental conditions. The biological effect of (1,3)-beta-D-glucans is a core issue stimulating large research efforts of the molecular properties and their consequences for action as biological response modifiers. The fascination for these molecules increased further following the finding of their ability to form complexes of defined geometry with a number of structures, ranging from linear architectures as polymers or carbon nanotubes, to globular structures as gold particles or dye molecules. The fascinating information concerning the relationship between sample treatment history and molecular organization has not yet reached out to all the contributors within the field, resulting in unnecessary apparent inconsistencies in the literature. In addition to environmental conditions, the sample history is known to influence on the precise structural organization of these molecules. The present knowledge related to the structure of native as well as denatured, renatured and annealed (1,3)-beta-D-glucans is reviewed. The influence of their structural organization on the biological activity and complexation abilities is discussed, and some factors hindering progress in the understanding of their biological effects or complexation abilities are pointed out.     

Anti-initiating activity of 3beta-methoxy-13alpha,14alpha-epoxyserratan-21beta-ol (PJJ-34) from the stem bark of Picea jezoensis Carr. var. jezoensis

Ultraviolet light is the major cause of skin cancers in human, and several effects of ultraviolet light B (UVB) are thought to contribute to skin photocarcinogenesis. 3Beta-methoxy-13alpha,14alpha-epoxyserratan-21beta-ol (PJJ-34; 1) isolated from Picea jezoensis Carr. var. jezoensis showed the strongest antitumor-promoting activity among naturally occurring triterpenoids in the in vivo two-stage mouse skin-carcinogenesis test. To investigate the anti-initiating activity, we further studied mouse models initiated with ultraviolet-B (UVB) and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). Oral administration of the PJJ-34 (1) during a period before and after the three times of UVB irradiation led to a remarkable effect: oral administration of a 0.0025% solution of 1 only to the test group, which started one week before and ended one week after the irradiation, showed less than half papillomas, and inhibition of tumor incidence and tumor multiplicity in comparison to the control group. Therefore, it was recognized that PJJ-34 (1) showed strong anti-initiating activity as well as anti-promoting activity. After all, 1 seems to be useful as cancer-chemopreventive agent.     

Differential in vivo effects of α‐naphthoflavone and β‐naphthoflavone on CYP1A1 and CYP2E1 in rat liver,lung, heart,and kidney

Male Sprague–Dawley rats were treated intraperitoneally with corn oil, the aryl hydrocarbon receptor (AHR) agonist β‐naphthoflavone (βNF), or the relatively weak AHR agonist α‐naphthoflavone (αNF). Animals treated with βNF experienced a significant loss (12%) of total body mass over 5 days and a dramatic elevation of CYP1A1 mRNA in all of the organs studied. Treatment with αNF had no significant effect on body mass after 5 days and caused only minor increases of liver, kidney, and heart CYP1A1 mRNA. In contrast, lung CYP1A1 mRNA was increased by αNF treatment to levels comparable to that seen with βNF treatment. CYP2E1 mRNA levels were also elevated in liver, lung, kidney, and heart in response to βNF treatment, whereas αNF was without effect. Large increases of CYP1A1‐dependent 7‐ethoxyresorufin O‐deethylation (EROD) activity occurred with microsomes prepared from the tissues of βNF‐treated animals. Comparatively small changes were associated with αNF treatment, with the exception of lung, where EROD activity was increased to approximately 60% of that with βNF treatment. CYP2E1‐dependent p‐nitrophenol hydroxylase (PNP) activity was also increased by βNF treatment in microsomes prepared from kidney (3.1‐fold), whereas αNF was without effect. In contrast, αNF or βNF treatment caused significant decreases of lung microsomal PNP (72% and 27% of corn oil control, respectively) and 7‐pentoxyresorufin O‐deethylation (48% and 17% of corn oil control, respectively) activities, indicating that PNP activity may be catalyzed by P450 isoforms other than CYP2E1 in rat lung. We conclude that βNF and αNF have differential effects on the expression and catalytic activity of CYP1A1 and CYP2E1, depending upon the organ studied. These changes most likely occur as a result of the direct actions of these compounds as AHR agonists, in addition to secondary effects associated with AHR‐mediated toxicity. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 29–40, 1999     

Studies on intramolecular hydrogen bonding between the pyridine nitrogen and the amide hydrogen of the peptide: synthesis and conformational analysis of tripeptides containing novel amino acids with a pyridine ring.

For the first time tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1) and AA(3) = Gly or Aib, Xaa = 2Pmg and 2Pyg) were prepared containing alpha-methyl-alpha-(2-pyridyl)glycine (2Pmg) and alpha-(2-pyridyl)glycine (2Pyg) by solid-phase Ugi reaction. These results clearly indicate that for the preparation of tripeptides containing an amino acid with a pyridine ring, the solid-phase Ugi reaction is very useful.NMR analysis clarified that 2Pmg-containing tripeptides adopt a unique conformation with an intramolecular hydrogen bond between 2Pmg-NH and the pyridine nitrogen. However, in the case of Z-Gly-2Pyg-Gly-OMe, the intramolecular hydrogen bonding between 2Pyg-NH and the pyridine nitrogen was not observed, whereas Z-Aib-2Pyg-Aib-OMe adopts a unique conformation with an intramolecular hydrogen bond between 2Pyg-NH and a pyridine nitrogen. Conformational analysis of the tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1), AA(3) = Gly or Aib, Xaa = alpha,alpha-di(2-pyridyl)glycine (2Dpy), alpha-phenyl-alpha-(2-pyridyl)glycine (2Ppg), 2Pmg and 2Pyg), clarified that when an alpha,alpha-disubstituted glycine with a 2-pyridyl group at an alpha-carbon atom is introduced into any peptide, an intramolecular hydrogen bond between a pyridine nitrogen and an amide proton is formed and conformational mobility of the peptide backbone is restricted.     

Mitogenic activity of particulate yeast β-(1 → 3)-d-glucan and its water-soluble derivatives

Particulate β-d-glucan was isolated from baker's yeast using autolysis and delipidization of the cells, followed by alkaline and acid treatment. The residual water-insoluble glucan termed cerevan has a β-(1→ 3)-linked backbone with β-(1 → 6)-linked short side chains. In order to achieve water solubility of the glucan, various derivatives were prepared (car☐ymethyl-, car☐yethyl-, hydroxyethyl-, sulfoethyl-), and the β-glucan was oxidized to glucuronoglucan. Their solubility, degree of substitution (DS), and molecular weight distribution (M_w) were compared. The immunomodulatory activity of these preparations was investigated in mitogenic and co-mitogenic tests on rat thymocytes. Cerevan showed higher stimulation indices compared with the known immunomodulator zymosan. Of the water-soluble derivatives, sulfoethylglucan was found to be the most active. Of the car☐ymethyl derivatives of various DS, the preparation with DS=0.75 exhibited the highest activity. Water-soluble car☐ymethyl preparations with DS > 1.0 and low-molecular-weight glucuronoglucan were inactive.     

ERK1/2 and Akt pathway activated during (3R,6R)-bassiatin(1)-induced apoptosis in MCF-7 cells

(3R,6R)-bassiatin(1) was isolated from the endogenous fungus, Fusarium oxysporum J8-1-2. Previous studies showed that (3R,6R)-bassiatin(1) has anti-oestrogen properties which make it cytotoxic to ER (oestrogen receptor)-positive breast cancer cells (MCF-7) by cell cycle arrest and induction of apoptosis. (3R,6R)-bassiatin(1) suppresses mRNA and protein expression of the ERα and oestrogen responsive genes of cyclin D1 and PR. We have investigated the interaction between (3R,6R)-bassiatin(1) and ERα and followed the roles of ERK (extracellular-signal-regulated kinase), Akt and GSK3β (glycogen synthase kinase 3β) during (3R,6R)-bassiatin(1)-induced apoptosis of MCF-7 cells. (3R,6R)-bassiatin(1) competed with E2 (17β-estradiol) for ERα active sites to inhibit ERα activation. However, while ERK1/2 and Akt were activated, GSK3β was inactivated during (3R,6R)-bassiatin(1)-induced apoptosis, suggesting that this compound is indeed an anti-oestrogen agent that can also activate the survival signalling pathway. Apoptosis caused by (3R,6R)-bassiatin(1) may be related to activation of ERK.