Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations

The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.DFG, 390540038, EXC 2008: UniSysCatDFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

Thermodynamic reaction control of nucleoside phosphorolysis

Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose‐1‐phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase‐catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate‐specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature‐dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis.DFG, 390540038, EXC 2008: UniSysCatTU Berlin, Open-Access-Mittel - 201

A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases

Efficient reaction monitoring is crucial for data acquisition in kinetic and mechanistic studies. However, for conversions of nucleosides to their corresponding nucleobases, as observed in enzymatically catalyzed nucleoside phosphorylation reactions, the current analytical arsenal does not meet modern requirements regarding cost, speed of analysis and high throughput. Herein, we present a UV/Vis spectroscopy-based assay employing an algorithm for spectral unmixing in a 96-well plate format. The algorithm relies on fitting of reference spectra of nucleosides and their bases to experimental spectra and allows determination of nucleoside/nucleobase ratios in solution with high precision. The experimental procedure includes appropriate dilution of a sample into aqueous alkaline solution, transfer to a multi-well plate, measurement of a UV/Vis spectrum and subsequent in silico spectral unmixing. This enables data collection in a high-throughput fashion and reduces costs compared to state-of-the-art HPLC analyses by approximately 5-fold while being 20-fold faster and offering comparable precision. Additionally, the method is robust regarding dilution and sample transfer errors as it only considers spectral form and not absolute intensity. It can be applied to all natural nucleosides and nucleobases and even unnatural ones as demonstrated by several examples.DFG, 390540038, EXC 2008: UniSysCa

AKT1 (E17K) mutation profiling in breast cancer: prevalence, concurrent oncogenic alterations, and blood-based detection.

BACKGROUND: The single hotspot mutation AKT1 [G49A:E17K] has been described in several cancers, with the highest incidence observed in breast cancer. However, its precise role in disease etiology remains unknown. METHODS: We analyzed more than 600 breast cancer tumor samples and circulating tumor DNA for AKT1 (E17K) and alterations in other cancer-associated genes using Beads, Emulsions, Amplification, and Magnetics digital polymerase chain reaction technology and targeted exome sequencing. RESULTS: Overall AKT1 (E17K) mutation prevalence was 6.3 % and not correlated with age or menopausal stage. AKT1 (E17K) mutation frequency tended to be lower in patients with grade 3 disease (1.9 %) compared with those with grade 1 (11.1 %) or grade 2 (6 %) disease. In two cohorts of patients with advanced metastatic disease, 98.0 % (n = 50) and 97.1 % (n = 35) concordance was obtained between tissue and blood samples for the AKT1 (E17K) mutation, and mutation capture rates of 66.7 % (2/3) and 85.7 % (6/7) in blood versus tissue samples were observed. Although AKT1-mutant tumor specimens were often found to harbor concurrent alterations in other driver genes, a subset of specimens harboring AKT1 (E17K) as the only known driver alteration was also identified. Initial follow-up survival data suggest that AKT1 (E17K) could be associated with increased mortality. These findings warrant additional long-term follow-up. CONCLUSIONS: The data suggest that AKT1 (E17K) is the most likely disease driver in certain breast cancer patients. Blood-based mutation detection is achievable in advanced-stage disease. These findings underpin the need for a further enhanced-precision medicine paradigm in the treatment of breast cancer

Women’s experiences of wearing therapeutic footwear in three European countries

Background: Therapeutic footwear is recommended for those people with severe foot problems associated with rheumatoid arthritis (RA). However, it is known that many do not wear them. Although previous European studies have recommended service and footwear design improvements, it is not known if services have improved or if this footwear meets the personal needs of people with RA. As an earlier study found that this footwear has more impact on women than males, this study explores women’s experiences of the process of being provided with it and wearing it. No previous work has compared women’s experiences of this footwear in different countries, therefore this study aimed to explore the potential differences between the UK, the Netherlands and Spain. Method: Women with RA and experience of wearing therapeutic footwear were purposively recruited. Ten women with RA were interviewed in each of the three countries. An interpretive phenomenological approach (IPA) was adopted during data collection and analysis. Conversational style interviews were used to collect the data. Results: Six themes were identified: feet being visibly different because of RA; the referring practitioners’ approach to the patient; the dispensing practitioners’ approach to the patient; the footwear being visible as different to others; footwear influencing social participation; and the women’s wishes for improved footwear services. Despite their nationality, these women revealed that therapeutic footwear invokes emotions of sadness, shame and anger and that it is often the final and symbolic marker of the effects of RA on self perception and their changed lives. This results in severe restriction of important activities, particularly those involving social participation. However, where a patient focussed approach was used, particularly by the practitioners in Spain and the Netherlands, the acceptance of this footwear was much more evident and there was less wastage as a result of the footwear being prescribed and then not worn. In the UK, the women were more likely to passively accept the footwear with the only choice being to reject it once it had been provided. All the women were vocal about what would improve their experiences and this centred on the consultation with both the referring practitioner and the practitioner that provides the footwear. Conclusion: This unique study, carried out in three countries has revealed emotive and personal accounts of what it is like to have an item of clothing replaced with an ‘intervention’. The participant’s experience of their consultations with practitioners has revealed the tension between the practitioners’ requirements and the women’s ‘social’ needs. Practitioners need greater understanding of the social and emotional consequences of using therapeutic footwear as an intervention

Impact of facial conformation on canine health: Brachycephalic Obstructive Airway Syndrome

The domestic dog may be the most morphologically diverse terrestrial mammalian species known to man; pedigree dogs are artificially selected for extreme aesthetics dictated by formal Breed Standards, and breed-related disorders linked to conformation are ubiquitous and diverse. Brachycephaly–foreshortening of the facial skeleton–is a discrete mutation that has been selected for in many popular dog breeds e.g. the Bulldog, Pug, and French Bulldog. A chronic, debilitating respiratory syndrome, whereby soft tissue blocks the airways, predominantly affects dogs with this conformation, and thus is labelled Brachycephalic Obstructive Airway Syndrome (BOAS). Despite the name of the syndrome, scientific evidence quantitatively linking brachycephaly with BOAS is lacking, but it could aid efforts to select for healthier conformations. Here we show, in (1) an exploratory study of 700 dogs of diverse breeds and conformations, and (2) a confirmatory study of 154 brachycephalic dogs, that BOAS risk increases sharply in a non-linear manner as relative muzzle length shortens. BOAS only occurred in dogs whose muzzles comprised less than half their cranial lengths. Thicker neck girths also increased BOAS risk in both populations: a risk factor for human sleep apnoea and not previously realised in dogs; and obesity was found to further increase BOAS risk. This study provides evidence that breeding for brachycephaly leads to an increased risk of BOAS in dogs, with risk increasing as the morphology becomes more exaggerated. As such, dog breeders and buyers should be aware of this risk when selecting dogs, and breeding organisations should actively discourage exaggeration of this high-risk conformation in breed standards and the show ring