MutationalPatterns: the one stop shop for the analysis of mutational processes

BACKGROUND: The collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data. RESULTS: Here, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights. CONCLUSIONS: This novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns

Characterization of Novel Antimalarial Compound ACT-451840: Preclinical Assessment of Activity and Dose-Efficacy Modeling.

BACKGROUND: Artemisinin resistance observed in Southeast Asia threatens the continued use of artemisinin-based combination therapy in endemic countries. Additionally, the diversity of chemical mode of action in the global portfolio of marketed antimalarials is extremely limited. Addressing the urgent need for the development of new antimalarials, a chemical class of potent antimalarial compounds with a novel mode of action was recently identified. Herein, the preclinical characterization of one of these compounds, ACT-451840, conducted in partnership with academic and industrial groups is presented. METHOD AND FINDINGS: The properties of ACT-451840 are described, including its spectrum of activities against multiple life cycle stages of the human malaria parasite Plasmodium falciparum (asexual and sexual) and Plasmodium vivax (asexual) as well as oral in vivo efficacies in two murine malaria models that permit infection with the human and the rodent parasites P. falciparum and Plasmodium berghei, respectively. In vitro, ACT-451840 showed a 50% inhibition concentration of 0.4 nM (standard deviation [SD]: ± 0.0 nM) against the drug-sensitive P. falciparum NF54 strain. The 90% effective doses in the in vivo efficacy models were 3.7 mg/kg against P. falciparum (95% confidence interval: 3.3-4.9 mg/kg) and 13 mg/kg against P. berghei (95% confidence interval: 11-16 mg/kg). ACT-451840 potently prevented male gamete formation from the gametocyte stage with a 50% inhibition concentration of 5.89 nM (SD: ± 1.80 nM) and dose-dependently blocked oocyst development in the mosquito with a 50% inhibitory concentration of 30 nM (range: 23-39). The compound's preclinical safety profile is presented and is in line with the published results of the first-in-man study in healthy male participants, in whom ACT-451840 was well tolerated. Pharmacokinetic/pharmacodynamic (PK/PD) modeling was applied using efficacy in the murine models (defined either as antimalarial activity or as survival) in relation to area under the concentration versus time curve (AUC), maximum observed plasma concentration (Cmax), and time above a threshold concentration. The determination of the dose-efficacy relationship of ACT-451840 under curative conditions in rodent malaria models allowed prediction of the human efficacious exposure. CONCLUSION: The dual activity of ACT-451840 against asexual and sexual stages of P. falciparum and the activity on P. vivax have the potential to meet the specific profile of a target compound that could replace the fast-acting artemisinin component and harbor additional gametocytocidal activity and, thereby, transmission-blocking properties. The fast parasite reduction ratio (PRR) and gametocytocidal effect of ACT-451840 were recently also confirmed in a clinical proof-of-concept (POC) study

Human and rat organ slices : a tool to study drug metabolism and toxicity

The research described in this thesis was aimed to develop a universal in vitro infrastructure to study drug metabolism and toxicity in liver, lung, kidney and intestines using precision-cut slices from both human and animal organs. Liver slices have been applied in numerous studies before. Also, lung and kidney slices have been used in several studies on drug metabolism and toxicity, but the use of intestinal slices was only scarcely described. ... Zie: Summary.

Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction

Animal models are commonly used in the preclinical development of new drugs to predict the metabolic behaviour of new compounds in humans. It is, however, important to realise that humans differ from animals with regards to isoform composition, expression and catalytic activities of drug-metabolising enzymes. in this review the authors describe similarities and differences in this respect among the different species, including man. This may be helpful for drug researchers to choose the most relevant animal species in which the metabolism of a compound can be studied for extrapolating the results to humans. The authors focus on CYPs, which are the main enzymes involved in numerous oxidative reactions and often play a critical role in the metabolism and pharmacokinetics of xenobiotics. In addition, induction and inhibition of CYPs are compared among species. The authors conclude that CYP2E1 shows no large differences between species, and extrapolation between species appears to hold quite well. In contrast, the species-specific isoforms of CYP1A, -2C, -2D and -3A show appreciable interspecies differences in terms of catalytic activity and some caution should be applied when extrapolating metabolism data from animal models to humans

Whole-genome sequencing and mutational analysis of human cord-blood derived stem and progenitor cells

Mutational signatures have been identified in cancer genomes, providing information about the causes of cancer and treatment vulnerabilities. This protocol describes an assay to determine the genotoxic mechanisms underlying these signatures using cord-blood derived hematopoietic stem and progenitor cells (CB-HSPCs). CB-HSPCs have a low mutation background, enabling sensitive detection of mutations. First, CB-HSPCs are exposed in vitro, sorted, and clonally expanded. This expansion enables whole-genome sequencing to detect the mutation load and respective patterns induced during genotoxic exposure. For complete details on the use and execution of this protocol, please refer to de Kanter et al. (2021)

Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies

Precision-cut tissue slices (PCTS) are viable ex vivo explants of tissue with a reproducible, well defined thickness. They represent a mini-model of the organ under study and contain all cells of the tissue in their natural environment, leaving intercellular and cell-matrix interactions intact, and are therefore highly appropriate for studying multicellular processes. PCTS are mainly used to study the metabolism and toxicity of xenobiotics, but they are suitable for many other purposes. Here we describe the protocols to prepare and incubate rat and human liver and intestinal slices. Slices are prepared from fresh liver by making a cylindrical core using a drill with a hollow bit, from which slices are cut with a specially designed tissue slicer. Intestinal tissue is embedded in cylinders of agarose before slicing. Slices remain viable for 24 h (intestine) and up to 96 h (liver) when incubated in 6- or 12-well plates under 95% O(2)/5% CO(2) atmosphere

Assessment of Drug Metabolism Enzyme and Transporter Pharmacogenetics in Drug Discovery and Early Development: Perspectives of the I-PWG

Genetic variants of drug metabolism enzymes and transporters can result in high pharmacokinetic and pharmacodynamics variability, unwanted characteristics of efficacious and safe drugs. Ideally, the contributions of these enzymes and transporters to drug disposition can be predicted from in vitro experiments and in silico modeling in discovery or early development, and then be utilized during clinical development. Recently, regulatory agencies have provided guidance on the preclinical investigation of pharmacogenetics, for application to clinical drug development. This white paper summarizes the results of an industry survey on current practice and challenges with using in vitro systems and in silico models to understand pharmacogenetic causes of variability in drug disposition