The Search for the Spatial and Electronic Requirements of a Drug

A new program called GAMMA (genetic algorithm for multiple molecule alignment) has been developed for the superimposition of several three-dimensional chemical structures. Superimposition of molecules and evaluation of structural similarity is an important task in drug design and pharmaceutical research. Similarities of compounds are determined by this program either based on their structural or their physicochemical properties by defining different matching criteria. These matching criteria are atomic properties such as atomic number or partial atomic charges. The program is based on a combination of a genetic algorithm with a numerical optimization process. A major goal of this hybrid procedure is to address the conformational flexibility of ligand molecules adequately. Thus, only one conformation per structure is necessary and the program can work even when only one conformation of a compound is stored in a database. The genetic algorithm optimizes in a nondeterministic process the size and the geometric fit of the overlay. The geometric fit of the conformations is further improved by changing torsional angles combining the genetic algorithm and the directed tweak method. The determination of the fitness of a superimposition is based on the Pareto optimization. As an application the superimposition of a set of Cytochrome P450c17 enzyme inhibitors has been performed.Electronic Supplementary Material available.     

Cloning of a functional 25‐hydroxyvitamin D‐1α‐hydroxylase in zebrafish (Danio rerio)

Activation of precursor 25‐hydroxyvitamin D₃ (25D) to hormonal 1,25‐dihydroxyvitamin D₃ (1,25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25‐hydroxyvitamin D‐1α‐hydroxylase (1α‐hydroxylase). To establish new models for assessing the physiological importance of the 1α‐hydroxylase‐25D‐axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α‐hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5–150 nM) or active 1,25D (0.1–10 nM) induced dose responsive expression (15–95‐fold) of the vitamin D‐target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full‐length zebrafish cyp27b1 cDNA was then generated using RACE and RT‐PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC‐8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than twofold induction of CYP24A1 mRNA expression and a 25‐fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography–mass spectrometry

A sensitive negative ion chemical ionization (NCI) gas chromatographic–mass spectrometric (GC–MS) method was modified for the quantitation of valproic acid (VPA) metabolites generated from in vitro cDNA-expressed human microsomal cytochrome P450 incubations. The use of the inherent soft ionization nature of electron-capture NCI to achieve high sensitivity enabled us to conduct kinetic studies using small amounts of recombinant human P450 enzymes. The assay is based on the selective ion monitoring of the intense [M−181] fragments of pentafluorobenzyl (PFB) esters in the NCI mode, and has the following features: (1) a micro-extraction procedure to isolate VPA metabolites from small incubation volumes (100 μl); (2) a second step derivatization with tert.-butyldimethylsilylating reagents to enhance sensitivity for hydroxylated metabolites; (3) a short run-time (<30 min) while maintaining full separation of 15 VPA metabolites by using a narrow-bore non-polar DB-1 column plus a new temperature gradient; and (4) good reproducibility and accuracy (intra- and inter-assay RSDs <15%, bias <15%) by using seven deuterated derivatives of analytes as internal standards. The derivatives of mono- and diunsaturated metabolites, like the parent drug, produced abundant [M−181]⁻ ions while the hydroxylated metabolites gave an ion at m/z of 273, corresponding to the [M−181]⁻ ion of the tert.-butyldimethylsilyl ethers. In conclusion, the GC–NCI-MS analysis of valproate metabolites provided us with a high resolution and sensitivity necessary to conduct metabolic and kinetic studies of valproic acid in small volume samples typical of the in vitro cDNA-expressed micro-incubation enzymatic systems.     

exo-Brevicomin biosynthetic pathway enzymes from the Mountain Pine Beetle,Dendroctonus ponderosae

exoBrevicomin (exo-7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octane) is an important semiochemical for a number of beetle species, including the highly destructive Mountain Pine Beetle (Dendroctonus ponderosae). It is also found in other insects and the African elephant. Despite its significance, very little is known about its biosynthesis. A recent microarray analysis implicated a small cluster of three D. ponderosae genes in exo-brevicomin biosynthesis, two of which had identifiable open reading frames (Aw et al., 2010; BMC Genomics 11:215). Here we report further expression profiling of two genes in that cluster and functional analysis of their recombinantly-produced enzymes. One encodes a short-chain dehydrogenase that used NAD(P)⁺ as a co-factor to catalyze the oxidation of (Z)-6-nonen-2-ol to (Z)-6-nonen-2-one. We therefore named the enzyme (Z)-6-nonen-2-ol dehydrogenase (ZnoDH). The other encodes the cytochrome P450, CYP6CR1, which epoxidized (Z)-6-nonen-2-one to 6,7-epoxynonan-2-one with very high specificity and substrate selectivity. Both the substrates and products of the two enzymes are intermediates in the exo-brevicomin biosynthetic pathway. Thus, ZnoDH and CYP6CR1 are enzymes that apparently catalyze the antepenultimate and penultimate steps in the exo-brevicomin biosynthetic pathway, respectively.     

Cytochrome P-450- and Peroxidase-Dependent Activation of Procarbazine and Iproniazid in Mammalian Cells

Metabolism of hydrazine derivatives, procarbazine and iproniazid, to reactive free radical intermediates has been studied using spin-trapping techniques in intact human promyelocytic leukemia (HL60) and mouse hepatic cell lines. While HL60 cells have been shown to contain both myeloperoxidase and cytochrome P-450 enzymes, the hepatic cell line shows only cytochrome P-450 activity. Both peroxidases and cytochrome P-450 have been reported to catalyze biotransformation of hydrazines. Procarbazine and iproniazid were rapidly metabolized in these cell lines to methyl and isopropyl radicals, respectively. However, in HL60 cells, procarbazine was metabolized by myeloperoxidase while iproniazid was metab olized mostly by the cytochrome P-450 system. In the hepatic cells, both of these compounds were metabolized by the P-450 system.     

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2^E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from α-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.     

Cytochromes P450 and drug resistance

Cytochromes P450 are the key enzymes for activating and inactivating many drugs, in particular anticancer drugs. Therefore, individual expression levels of cytochromes P450 may play a crucial role in drug safety and drug efficacy. Overexpression of cytochrome P450 may yield rapid turnover and elimination of drugs before the target site was reached and any pharmacological effect is observed. Therefore, it may be vital to know the individual cytochrome P450 status in order to select the appropriate drug before drug resistance occurs. Expression levels and activity of cytochromes P450 depend on many different factors. These factors include tissue and organ specific expression, sex- and age-dependent expression, genetic differences yielding polymorphic forms, competitive inhibition or induction of cytochromes P450 due to multiple drug interaction, nutrition and diet. Genetically engineered test cells defined for cytochromes P450 are available for studying drugs for metabolic activation and for identifying the metabolically competent cytochrome P450 isoform.     

离体鼠肝细胞及P-450酶系与拟除虫菊酯类杀虫剂的相互作用

经研究表明顺式氯氰菊酯、甲氰菊酯、氟氰菊酯、氰戊菊酯在正辛醇——水相中的分配系数(logP)分别为6.13,5.50,5.55和4.02。离体培养的大白鼠原代肝细胞对上述杀虫剂(除氟氰菊酯外)的吸收系数(q值)分别为322.6,281.1,201.9。上述杀虫剂在离体培养的原代肝细胞中降解很快,温育30分钟后降解速率分别为90.0％(顺式氯氰菊酯)、86.2％(甲氰菊酯)、69.5％(氰戊菊酯)。同时有明显的减少离体培养的鼠肝细胞中P-450含量的作用,其中以溴氰菊酯和顺式氯氰菊酯作用较明显。在溴氰菊酯处理组中,随杀虫剂浓度的升高,这种减少P-450含量的作用明显增强。检查温育反应90分种后的细胞悬浮反应液,对照组仍维持高的细胞存活率(74.4％和91.2％),而杀虫剂处理组,除甲氰菊酯处理组仍有42.4％的存活率外,其余各杀虫剂(溴氰菊酯各浓度组和顺式氯氰菊酯处理组)均未检出存活的细胞。结果表明,试验所用的几种拟除虫菊酯类杀虫剂具有一定的细胞毒性。它们在鼠肝细胞中的降解代谢可能主要不是由于多功能氧化酶起作用。另外,杀虫剂的亲脂性与q值及在肝细胞中的降解速率三者间有一定的正相关。     

Changes in immunohistochemical distribution of cytochrome MC-P-448 in the rat liver during acclimation to cold

Immunohistochemical localization of cytochrome MC-P-448 (form of cytochrome P-450 induced by methylcholanthrene) in rat hepatic lobule and the changes in their distribution pattern in response to cold exposure at 4°C were investigated. The distribution of hepatocytes expressing immunoreactivity to cytochrome MC-P-448 was demonstrated with rabbit anti-MC-P-448 serum using a microphotomeasurement system P1 (Nikon). A duration of cold exposure for 1, 2, 3 or 4 weeks at 4°C was applied to study the effect of cold adaptation of cytochrome MC-P-448. In control rats housed at 24°C, hepatocytes showing high immunoreactivity to cytochrome MC-P-448 were located in the centrilobular areas of the hepatic lobules, whereas they disappeared markedly in the 4-week cold-exposed rats. In 1-week and 2-week cold-exposed rats, only a slight decrease in the expression of MC-P-448 positive hepatocytes was observed. These changes were clearly seen by visual inspection of the distribution topography as determined by a microphotomeasurement technique. In conclusion, cytochrome MC-P-448 forms which were predominantly located in centrilobular areas in the hepatic lobule decreased in 4-week cold-exposed rats. This was in contrast to our early report which showed an increasing tendency of cytochrome PB-P-450 forms in 4-week cold-exposed rats.     

Thiomorpholine and morpholine oxidation by a cytochrome P450 in Mycobacterium aurum MO1. Evidence of the intermediates by in situ 1H NMR

Spectrophotometric assays of Mycobacterium aurum MO1 cells extracts gave evidence of a soluble cytochrome P450, involved in the degradative pathway of morpholine, a waste product from the chemical industry. In order to get further information, the kinetics of the biodegradation of the sulfur analogue thiomorpholine was monitored by using in situ nuclear magnetic resonance (NMR). This technique allowed the identification of two intermediates: the sulfoxide of thiomorpholine resulting from S-oxidation and thiodiglycolic acid owing to ring cleavage. The S-oxidation (S SO) represents one of the well-known reactions catalyzed by cytochromes P450. The inhibitory effect of metyrapone, a cytochrome P450 inhibitor, on the thiomorpholine and morpholine degradative abilities of M. aurum MO1 confirmed the involvement of a cytochrome P450. These results and the decrease of the rate of formation of the first intermediate during the morpholine degradation, 2-(2-aminoethoxy) acetate, proved the key role of the cytochrome P450 in the early events of the biodegradation, i.e, in the C–-N bond cleavage.