Quantification of Normal Cell Fraction and Copy Number Neutral LOH in Clinical Lung Cancer Samples Using SNP Array Data

Technologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells.Using SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection.Genome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes

Reducing Attrition via Improved Strategies for Pre-clinical Drug Discovery : SPR-biosensor Aided Interaction Studies

The efficacy of a drug is tightly intertwined with its interaction mechanism with the drug target. The mechanism is dependent on the physicochemical and structural characteristics of both target and drug molecule. Drug discovery is plagued by a high attrition rate, whereas in the clinic, a major issue is drug resistance. To improve the quality of the lead compounds in the pre-clinical phase of drug discovery, and thereby reducing the attrition, a deeper understanding of interaction mechanisms is needed. We have adopted new strategies and techniques for this purpose. A compound library was compiled for the purpose of fragment-based drug discovery. Its compatibility with the SPR platform, along with its interaction profile, was validated. The library was subsequently used in a screening campaign for novel scaffolds of human immunodeficiency virus-1 protease, not sensitive to common resistance mutations. This was achieved by the use of a target panel containing signature resistance mutations towards already approved HIV-1 protease inhibitors. 10 scaffolds were identified and deemed novel. These constitute interesting starting points for development of a new generation of HIV-1 protease inhibitors with different resistance mechanisms, which is very valuable in combination therapies. The cause of difference in anti-viral potency in cell cultures was investigated for two iso-affinity compounds acting on the hepatitis C viral polymerase, NS5B. By SPR-aided interaction analysis with chemo- and thermodynamic characterization, filibuvir and VX-222, both same-site allosteric inhibitors in phase II clinical trials, were identified to have two different interaction mechanisms. This was ultimately suggested to cause the differences in potency. A structure-kinetic relationship study, with a thermodynamic characterization, was performed for an approved thrombin inhibitor and five close P3-analogues. This study had the aim to better understand the basic mechanisms of the interactions. Stopped-flow spectroscopy, SPR, and calorimetry were used in parallel and their results compared before evaluation with x-ray crystallography data. Thus, this thesis has demonstrated successful use of fragment-based drug discovery and high resolution techniques to advance projects in most stages of pre-clinical drug discovery with the aim to reduce the future drug attrition and to understand molecular interactions on a fundamental level

Structure-based discovery of pyrazolobenzothiazine derivatives as inhibitors of hepatitis C virus replication

The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. To identify novel structural hits as anti-HCV agents, we performed structure-based virtual screening of our in-house library followed by rational drug design, organic synthesis, and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the meta-fluoro-N-1-phenyl pyrazolobenzothiazine derivative 4a, which exhibited an EC50 = 3.6 μM, EC90 = 25.6 μM, and CC50 > 180 μM in the Huh 9-13 replicon system, thus providing a good starting point for further hit evolution.status: publishe

The DNA damage response activates HPV16 late gene expression at the level of RNA processing

We show that the alkylating cancer drug melphalan activated the DNA damage response and induced human papillomavirus type 16 (HPV16) late gene expression in an ATM- and Chk1/2-dependent manner. Activation of HPV16 late gene expression included inhibition of the HPV16 early polyadenylation signal that resulted in read-through into the late region of HPV16. This was followed by activation of the exclusively late, HPV16 splice sites SD3632 and SA5639 and production of spliced late L1 mRNAs. Altered HPV16 mRNA processing was paralleled by increased association of phosphorylated BRCA1, BARD1, BCLAF1 and TRAP150 with HPV16 DNA, and increased association of RNA processing factors U2AF65 and hnRNP C with HPV16 mRNAs. These RNA processing factors inhibited HPV16 early polyadenylation and enhanced HPV16 late mRNA splicing, thereby activating HPV16 late gene expression

The versatile nature of the 6-aminoquinolone scaffold: identification of submicromolar hepatitis C virus NS5B inhibitors

We have previously reported that the 6-aminoquinolone chemotype is a privileged scaffold to obtain antibacterial and antiviral agents. Herein we describe the design, synthesis, and enzymatic and cellular characterization of new 6-aminoquinolone derivatives as potent inhibitors of NS5B polymerase, an attractive and viable therapeutic target to develop safe anti-HCV agents. The 6-amino-7-[4-(2-pyridinyl)-1-piperazinyl]quinolone derivative 8 proved to be the best compound of this series, exhibiting an IC50 value of 0.069 μM against NS5B polymerase and selective antiviral effect (EC50 = 3.03 μM) coupled with the absence of any cytostatic effect (CC50 > 163 μM; SI > 54) in Huh 9-13 cells carrying a HCV genotype 1b, as measured by MTS assay. These results indicate that the 6-aminoquinolone scaffold is worthy of further investigation in the context of NS5B-targeted HCV drug discovery programs.status: publishe

The versatile nature of the 6-aminoquinolone scaffold: identification of submicromolar hepatitis C virus NS5B inhibitors

We have previously reported that the 6-aminoquinolone chemotype is a privileged scaffold to obtain antibacterial and antiviral agents. Herein we describe the design, synthesis, enzymatic and cellular characterization of new 6-aminoquinolone derivatives as potent inhibitors of NS5B polymerase, an attractive and viable therapeutic target to develop safe anti-HCV agents. The 6-amino-7-[4-(2-pyridinyl)-1-piperazinyl] quinolone derivative 8 proved to be the best compound of this series, exhibiting IC50 value of 0.069 μM against NS5B polymerase and selective antiviral effect (EC50 = 3.03 µM, EC90 =13.5 μM) coupled with the absence of any cytostatic effect (CC50 >163 µM, SI >54) in a HCV subgenomic replicon system. These results clearly indicate that the 6-aminoquinolone scaffold is worthy of further investigation in the context of NS5B-targeted HCV drug discovery programs

Structure-Based Discovery of Pyrazolobenzothiazine Derivatives As Inhibitors of Hepatitis C Virus Replication

The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. To identify novel structural hits as anti-HCV agents, we performed structure-based virtual screening of our in-house library followed by rational drug design, organic synthesis, and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the <i>meta</i>-fluoro-<i>N</i>-1-phenyl pyrazolobenzothiazine derivative <b>4a</b>, which exhibited an EC₅₀ = 3.6 μM, EC₉₀ = 25.6 μM, and CC₅₀ > 180 μM in the Huh 9–13 replicon system, thus providing a good starting point for further hit evolution

Active Site Mapping of an Aspartic Protease by Multiple Fragment Crystal Structures: Versatile Warheads To Address a Catalytic Dyad

Crystallography is frequently used as follow-up method to validate hits identified by biophysical screening cascades. The capacity of crystallography to directly screen fragment libraries is often underestimated, due to its supposed low-throughput and need for high-quality crystals. We applied crystallographic fragment screening to map the protein-binding site of the aspartic protease endothiapepsin by individual soaking experiments. Here, we report on 41 fragments binding to the catalytic dyad and adjacent specificity pockets. The analysis identifies already known warheads but also reveals hydrazide, pyrazole, or carboxylic acid fragments as novel functional groups binding to the dyad. A remarkable swapping of the S1 and S1′ pocket between structurally related fragments is explained by either steric demand, required displacement of a well-bound water molecule, or changes of trigonal-planar to tetrahedral geometry of an oxygen functional group in a side chain. Some warheads simultaneously occupying both S1 and S1′ are promising starting points for fragment-growing strategies