Therapeutic targeting of ATR in alveolar rhabdomyosarcoma

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS

A Nazarov‐Ene Tandem Reaction for the Stereoselective Construction of Spiro Compounds

The different reactivity of trienones under Lewis and Brønsted acids catalysis was investigated, resulting in distinct cyclization products and carbon backbones that originated either from a conjugate Prins cyclization or an interrupted Nazarov cyclization. In particular, an unprecedented Nazarov cyclization tandem reaction is presented, terminating the oxyallyl cation by an ene-type reaction, and leading stereoselectively to bicyclic spiro compounds. The terminal olefin of this motif represents a useful handle for further functionalization, making it a strategic intermediate in total syntheses. The tandem Nazarov/ene cyclization was shown to be preferred over a Nazarov/[3+2] tandem reaction for all our substrates, independent of chain length. Deuteration studies further support the mechanistic hypothesis of the terminating ene reaction

Development of a countergradient parking system for gradient liquid chromatography with online biochemical detection of serine protease inhibitors

A gradient HPLC approach in combination with a countergradient system for online biochemical detection (BCD) to screen for inhibitors of serine proteases is described. For gradient separations, this novel countergradient system was developed to produce a biocompatible constant solvent composition in the BCD. The countergradient system is based on retaining complete gradients in an additional preparative HPLC column, followed by subsequent and reversible elution to the separation column effluent. Major advantages compared with existing countergradient systems are that no additional LC pumps are needed and enhanced stability. The developed countergradient system was systematically characterized applying different gradient programs. Inhibitors eluting in a post-column continuous flow analysis interfere with the enzymatic release of fluorescent 7-amino-4-methylcoumarin (AMC) from an AMC-labeled peptide. The inhibitory activity of eluting substances is sensitively detected as the degree of reduced fluorescence intensity. This biochemical detection system (BCD) for proteases was validated with three known inhibitors of the benzamidine type. Their I

Digital microfluidic sample preparation for biological mass spectrometry

The use of mass spectrometry in the biosciences has undergone huge growth in re- cent years due to sustained effort in the development of new ionisation techniques, more powerful mass analysers and better bioinformatic tools. These developments mean that it is now possible to introduce complex crude biological-mixtures into a mass spectrometric platform and to obtain detailed information about the sample. The front-end sample handling techniques used for sample preparation have, for the most part, not changed despite the recent advances in hyphenation of liquid- chromatography and mass spectrometry required to tackle the issue of increased sample complexity. In this thesis the possibility of using Digital Microfluidics (DMF) for front-end sample preparation prior to mass-spectrometric analysis of protein samples has been investigated. DMF is a micro-electromechanical system (MEMS) technology used for manipulation of sub-microlitre droplets. The movement of discrete droplets of liquid is exploited using the Coulombic forces arising due to free charge polarisation. Droplets can be split, joined, dispensed and moved over a sub-surface electrode array. In this thesis a range of DMF devices have been designed, manufactured and coupled with mass spectrometric platforms for protein analysis. A variety of techniques for mass spectrometry- based analysis of biological samples from the fluidic chips have been investigated. A robotic system has been developed to automate sample introduction, manipulation and removal. Finally the application of on-chip sample purification and enzymatic digestion have been demonstrated, providing proof of concept for digital microfluidic sample preparation in mass spectrometry-based proteomics

Online screening of homogeneous catalyst performance using reaction detection mass spectrometry

An integrated online screening system was developed to rapidly screen homogeneous catalysts for activity toward a selected synthesis. The continuous-flow system comprises standard HPLC pumps for the delivery of substrates, an HPLC autosampler for the injection of homogeneous catalysts, a thermostated reactor to mediate synthesis, and a single-stage quadrupole mass spectrometer (MS) equipped with atmospheric pressure chemical ionization for the determination of product formation. MS detection offers sensitivity, specificity, and speed when applied to the analysis of dynamic processes in the condensed phase. By applying the present methodology for the study of substrate conversion mediated by homogeneous catalysts, the concentration of substrates and reaction product could be monitored while information about the catalysts could also be obtained. In an initial screening application, the performance of a selected number of Lewis acids in the multicomponent synthesis of a highly substituted 2-imidazoline was determined. Limit of detection and limit of quantitation were determined by injecting different concentrations of 2-imidazoline standards and proved to be 1.6 and 5.2 nM, respectively. The results obtained with the new screening method were in good agreement with a traditional bench-scale experiment. Moreover, the system was capable of determining catalyst performance with very low catalyst and solvent consumption while the ruggedness of the system was exhibited with a 24-h continuous analysis of 280 successive catalyst injections with a peak area variation within 7% relative standard deviation. © 2008 American Chemical Society