Withanolides: Elucidating steroidal lactone biosynthesis in Nightshades

Withania somnifera (Solanaceae) is well known in ayurvedic medicine as a strengthening tonic for various medical purposes. Its effects are mainly due to withanolides, a class of steroidal lactones with diverse oxidation patterns present in various nightshade plants. Pharmacological studies attributed anti-proliferative and anti-inflammatory properties to withanolides. However, obtaining medicinally active withanolides can be complicated, as complex mixtures are present in producing plants and total synthesis of withanolides is costly and time consuming. Therefore, investigation of their biosynthesis is important to enable biotechnological enhancement and to provide novel insights into plant steroid biochemistry. This work aimed to investigate withanolide biosynthesis in Physalis peruviana and Withania somnifera. Both plants were investigated for their main withanolides, as producers can accumulate a diverse array of withanolides, depending on the cultivation conditions. Here, besides several known withanolides, two yet unknown, truncated withanolides (irinan A (1) and B (2)) were isolated from P. peruviana and their structures were elucidated. As intermediates of withanolide biosynthesis were needed for enzyme assays but are neither known, nor commercially available, metabolic engineering in yeast was attempted to divert yeast ergosterol biosynthesis towards production of 24-methyldesmosterol (3), the last known precursor in withanolide biosynthesis. However, while production of the precursor 24-methylenecholesterol (4) was temporarily observed, 3 did not accumulate. Furthermore, based on the biosynthetic hypothesis, 21 cytochrome P450 (P450) and 14 dehydratase (DH) gene candidates were selected after analysis of three withanolide-producing species. Of those, 17 P450 and 6 DH candidates could be cloned and evaluated by gene silencing in W. somnifera, identifying 5 P450 and 2 DH gene candidates where silencing evoked significant decrease of the main withanolide (withaferin A, 5). Those candidates were further examined by heterologous expression experiments in the model plant Nicotiana benthamiana. Here activity on the substrate 24-methyldesmosterol was detected for one candidate (P450-7), while another exhibited activity on native cycloartenol (6) from the host plant (P450-17). Further investigation of P450-17 revealed that orthologs were present in tomato and potato, both non-producers of withanolides. In both plants P450-17 homologous genes are arranged in gene clusters, with neither the genes nor the cluster being reported before. In conclusion, this work provides insights into oxidations involved in withanolide biosynthesis and yet unknown phytosterol pathways in Solanaceae plants

Formation mechanisms and aroma potential of 2-aminoacetophenone in boar fat

For decades the surgical castration of male piglets has been a common practice to avoid the incidence of boar taint within Europe. Boar taint is known as an offensive off-flavour occurring when meat of uncastrated boars is heated prior to consumption. According to the current state of knowledge the off-flavour’s perception by sensitive consumers is mainly caused by high endogenous concentrations of two substances: The boar pheromone androstenone (5α-androst-16-en-3-one) and the heterocyclic amine skatole (3-methylindole). As the surgical castration of male piglets without anaesthesia is known to cause serious pain and distress for the animals this practice is increasingly questioned and refused by the majority of consumers. Driven by this public concerns, in 2010 all European stakeholders along the production chain of pork voluntarily agreed to ban surgical castration without anaesthesia by the beginning of 2018 and search for alternative strategies to prevent consumers from tainting pork. Since the formulation of this declaration known as “declaration of Brussels” many efforts have been undertaken in order to minimize the levels of androstenone and skatole in boars, i.e. immunocastration, boar fattening. However, frequent discrepancies between the sensory evaluation of boar fat and analytically determined levels of androstenone and skatole raised the question whether and to what extend other substances contribute to the off-flavour. In this context compounds of miscellaneous classes have been discussed. Indole, like skatole a degradation product of L-tryptophan in the pig’s large intestine, and reduced metabolization products of androstenone, 5α-androsten-ol and 5β-androsten-ol, were proven to have a minor impact on the occurrence of boar taint. Furthermore, fatty acids and their microbiological degradation products as well as phenolic compounds were suggested as additional boar taint agents. Interestingly, no focus was put on hepatic phase-I-metabolites of skatole although seven products of the hepatic clearance of the amine were already identified almost 20 years ago. Among these metabolites a substance appears, that already attracted attention as a key substance within the origination of off-flavours in white wines and milk products, 2-aminoacetophenone. However, no data about 2-aminoacetophenone levels in boars exist and it’s biogenesis in pigs has not yet been elucidated. Thus, the presented thesis addressed the question of a possible contribution of 2-aminoacetophenone to the perception of boar taint and its formation in pigs

Molecular investigations of plant cytochrome P450

Purification of plant cyt P450s has been difficult, due to their general low abundance in plant tissues and instability during purification. This has meant that only a few have been directly purified from plant tissue to date. Thus, conventional methods for studying their catalytic activity, such as cloning procedures involving the use of antibodies, or the generation of oligonucleotide probes from amino acid sequence, have proven of limited success. An alternative molecular approach was therefore adopted to study cyt P450s from Zea mays and Arabidopsis. For Z. mays, degenerate PCR primers directed to two conserved regions of sequence were used to amplify a cyt P450 fragment with greatest homology to the CYP71 family of plant cyt P450s. Despite extensive screening of phage Z. mays leaf, safener-treated root and seedling cDNA libraries, a full-length clone was not isolated using this fragment as a probe. Genomic southern analysis confirmed that a gene corresponding to the cyt P450 fragment was part of the Z. mays genome. Northern analysis and reverse transcriptase-polymerase chain reaction indicated that it represented a transcript of low abundance, was present in shoot and root tissues and was constitutive with respect to age and treatments known to induce cyt P450s capable of herbicide metabolism. For Arabidopsis, an Expressed Sequence Tag (EST) from the Michigan State University EST programme, previously identified as a potential full-length cyt P450, was obtained for further analysis in an effort to assign function. Detailed sequence analysis confirmed it was a full-length cyt P450 of the CYP71 family, and it was designated CYP71B7. Northern analysis revealed it was expressed most strongly in rosette leaves and was also present in roots, leaves, siliques and flowers. Southern analysis indicated that it represented a single copy gene in the Arabidopsis genome. CYP71B7 was successfully expressed in Saccharomyces cerevisiae and biochemical analysis revealed it was active in ethoxycoumarin O-deethylation. 7-ethoxycoumarin is a general artificial substrate of cyt P450s. Several natural compounds were identified which inhibited this activity, and thus are candidate physiological substrates of CYP71B7

Modeling Chemical Interaction Profiles: II. Molecular Docking, Spectral Data-Activity Relationship, and Structure-Activity Relationship Models for Potent and Weak Inhibitors of Cytochrome P450 CYP3A4 Isozyme

Polypharmacy increasingly has become a topic of public health concern, particularly as the U.S. population ages. Drug labels often contain insufficient information to enable the clinician to safely use multiple drugs. Because many of the drugs are bio-transformed by cytochrome P450 (CYP) enzymes, inhibition of CYP activity has long been associated with potentially adverse health effects. In an attempt to reduce the uncertainty pertaining to CYP-mediated drug-drug/chemical interactions, an interagency collaborative group developed a consensus approach to prioritizing information concerning CYP inhibition. The consensus involved computational molecular docking, spectral data-activity relationship (SDAR), and structure-activity relationship (SAR) models that addressed the clinical potency of CYP inhibition. The models were built upon chemicals that were categorized as either potent or weak inhibitors of the CYP3A4 isozyme. The categorization was carried out using information from clinical trials because currently available in vitro high-throughput screening data were not fully representative of the in vivo potency of inhibition. During categorization it was found that compounds, which break the Lipinski rule of five by molecular weight, were about twice more likely to be inhibitors of CYP3A4 compared to those, which obey the rule. Similarly, among inhibitors that break the rule, potent inhibitors were 2–3 times more frequent. The molecular docking classification relied on logistic regression, by which the docking scores from different docking algorithms, CYP3A4 three-dimensional structures, and binding sites on them were combined in a unified probabilistic model. The SDAR models employed a multiple linear regression approach applied to binned 1D 13C-NMR and 1D 15N-NMR spectral descriptors. Structure-based and physical-chemical descriptors were used as the basis for developing SAR models by the decision forest method. Thirty-three potent inhibitors and 88 weak inhibitors of CYP3A4 were used to train the models. Using these models, a synthetic majority rules consensus classifier was implemented, while the confidence of estimation was assigned following the percent agreement strategy. The classifier was applied to a testing set of 120 inhibitors not included in the development of the models. Five compounds of the test set, including known strong inhibitors dalfopristin and tioconazole, were classified as probable potent inhibitors of CYP3A4. Other known strong inhibitors, such as lopinavir, oltipraz, quercetin, raloxifene, and troglitazone, were among 18 compounds classified as plausible potent inhibitors of CYP3A4. The consensus estimation of inhibition potency is expected to aid in the nomination of pharmaceuticals, dietary supplements, environmental pollutants, and occupational and other chemicals for in-depth evaluation of the CYP3A4 inhibitory activity. It may serve also as an estimate of chemical interactions via CYP3A4 metabolic pharmacokinetic pathways occurring through polypharmacy and nutritional and environmental exposures to chemical mixtures

Application of phenotyping and therapeutic drug monitoring in personalised drug treatment : Peter Benjamin Berger aus Basel, Basel-Stadt

As every individual is a product of his/her genes and environment, which can be regarded as a paraphrase of an individual’s phenotypic characteristic, it becomes apparent as to why the concept of personalised medicine, i.e. tailoring a treatment regimen to an individual’s needs, remains an ever-prevalent topic in the medical community. Human cytochrome P450 enzymes (CYPs) are accountable for the oxidative metabolism of approximately 50% of commonly used drugs as well as endogenous compounds. However, CYP activity is exceedingly variable amongst individuals. Over the course of my PhD project, I worked on five different projects that implicated the utility of two valuable tools in personalised medicine: namely, phenotyping and therapeutic drug monitoring. The determination of a person’s enzymatic activity through phenotyping can help guide a GP’s effort to personalise drug therapy by administering the applicable dose, thereby improving efficacy and reducing side effects at the start of therapy. Therapeutic drug monitoring (TDM) on the other hand, enables the continued observation of a patient’s drug concentration, predominantly in plasma, thus allowing patients that are at risk of either over-/ or underdosing to be identified. In the past two decades, many different in vivo phenotyping cocktails, enabling the simultaneous assessment of multiple CYP isoforms, e.g. Cooperstown- [1], Inje- [2], Quebec- [3], Karolinska- [4], and the Pittsburgh-cocktail [5], as well as a great number of in vitro cocktails [6-14] were developed. Metabolism studies performed in vitro are useful to the extent of acquiring anticipatory information of in vivo predictions of CYP- inhibition/ -induction in an efficient time and cost saving manner. It is, however, uncommon to use established in vivo cocktails for in vitro studies [7]. Nevertheless, using probe substrates previously unaccustomed to in vitro cocktail studies, e.g. efavirenz (CYP2B6), losartan (CYP2C9), and metoprolol (CYP2D6), we were able to show the potential of the Basel cocktail [15, 16] to characterise a variety of different liver cell models. Of particular interest to our study was the characterisation of 3D primary human hepatocytes (PHH), co-cultured with 3T3-mouse fibroblasts [17]. By using the same batch of PHH in 2D and 3D-culture, we were able to show the functional benefits of a co-culture system, enabling hepatocytes to reside in a 3D environment, and leading to improved CYP activity and mRNA expression. In subsequent studies, we were able to provide explanations for pending in vivo observations [15], through combining knowledge of freshly acquired in vitro and in vivo characterisation data of the Basel cocktail. In the second study, we demonstrated that the α-hydroxymetoprolol formation is not mediated solely by CYP2D6, since under induced conditions and through experiments in isoform specific supersomes, the involvement of CYP3A4 also became apparent. This does not, however, impede the continued applicability of metoprolol as probe substrate of CYP2D6 since the involvement of CYP3A4 only becomes apparent when induction is evaluated, which is commonly not done for CYP2D6. In the third study, we were able to show, through an in vitro interactions study, that flurbiprofen can be used to replace losartan as a phenotyping drug for CYP2C9. Subsequently, a pilot study (n=2) showed the prospect of simplifying the cocktail administration through the use of a combi-capsule containing all six probe drugs of the modified Basel cocktail. After having previously tested the phenotyping capacities of the Basel cocktail in two studies involving young, healthy, male volunteers [15, 16], the fourth study of this thesis demonstrates that we successfully used caffeine (CYP1A2) and midazolam (CYP3A4) to phenotype elderly, patients of both sexes that were being treated for non-small cell lung cancer (NSCLC) with erlotinib (Tarceva®). In so doing, we were able to show that subjects with a slow CYP3A4 metabolism had a higher likelihood of developing cutaneous toxicity than patients with an extensive metabolism. While the fourth study touched upon the possible usefulness of collecting DBS with which to perform TDM, as opposed to conventional plasma samples, the fifth and final study was devoted entirely to this topic. Here, we demonstrated the development, validation, and application of an automated DBS extraction method, while incorporating an evaluation of an antiretroviral adherence/ therapeutic drug monitoring study. Our study was able to show that concentrations of two antiretroviral drugs, nevirapine and efavirenz, could reliably be determined by automated extraction in DBS samples that had been obtained in a challenging setting in rural Tanzania

Computer modeling of dapsone-mediated heteroactivation of flurbiprofen metabolism by CYP2C9

The occurrence of atypical kinetics in cytochrome P450 reactions can confound in vitro determinations of a drug\u27s kinetic parameters. During drug development, inaccurate kinetic parameter estimates can lead to incorrect decisions about a lead compound\u27s potential for success. It has become widely accepted that in certain CYP subfamilies more than one molecule can occupy the active site simultaneously, in some cases resulting in enhanced substrate turnover (heteroactivation). However, the specific mechanism(s) by which dual-compound binding results in heteroactivation remain unclear. It is known that orientation of the substrate in the active site, as dictated by interactions with active site residues, plays a large role in metabolic outcome. Effector compounds have been shown in vitro to alter substrate position in the active site. Here, data obtained via in silico methods including docking, molecular dynamics, semi-empirical and ab initio quantum mechanics indicate that direct interaction between effector and substrate can play a role in stabilizing the substrate in an alternative conformation conducive to oxidation. In this study a high-throughput screening computer model of heteroactivation of flurbiprofen metabolism by CYP2C9 has been developed for the purpose of elucidating key interactions between substrate, effector, and enzyme responsible for heteroactivation in this system, as well as to predict as yet unknown activators