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Nisha Kurian,1 Taylor S Cohen,2 Lisa Öberg,3 Erica De Zan,3 Gabriel Skogberg,4 Stefan Vollmer,3 Engin Baturcam,3 Petter Svanberg,5 Britta Bonn,5 Paul D Smith,6 Outi Vaarala,3 Danen M Cunoosamy3 1Respiratory Inflammation and Autoimmune (RIA) Precision Medicine Unit, Precision Medicine, Oncology R&D, AstraZeneca, Gothenburg, Sweden; 2Microbial Sciences, Medimmune, Gaithersburg, MD, USA; 3Translational Science and Experimental Medicine, Research and Early Development, RIA, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 4Bioscience, Research and Early Development, RIA, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 5Drug Metabolism and Pharmacokinetics, Research and Early Development, RIA, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 6Bioscience, Oncology R&D, AstraZeneca, Cambridge, UKCorrespondence: Nisha KurianRespiratory Inflammation and Autoimmune (RIA) Precision Medicine Unit, Precision Medicine, Oncology R&D, AstraZeneca, Gothenburg, SwedenTel +46 72 197 9662Email Nisha.e.kurian@astrazeneca.comBackground: Unlike p38 mitogen-activated protein Kinases (MAPK) that has been extensively studied in the context of lung-associated pathologies in COPD, the role of the dual-specificity mitogen-activated protein kinase kinase (MEK1/2) or its downstream signaling molecule extracellular signal-regulated kinases 1/2 (ERK1/2) in COPD is poorly understood.Objectives: The aim of this study was to address whether MEK1/2 pathway activation is linked to COPD and that targeting this pathway can improve lung inflammation through decreased immune-mediated inflammatory responses without compromising bacterial clearance.Methods: Association of MEK1/2 pathway activation to COPD was investigated by immunohistochemistry using lung tissue biopsies from COPD and healthy individuals and through analysis of sputum gene expression data from COPD patients. The anti-inflammatory effect of MEK1/2 inhibition was assessed on cytokine release from lipopolysaccharide-stimulated alveolar macrophages. The effect of MEK1/2 inhibition on bacterial clearance was assessed using Staphylococcus aureus killing assays with RAW 264.7 macrophage cell line and human neutrophils.Results: We report here MEK1/2 pathway activation demonstrated by increased pERK1/2 staining in bronchial epithelium and by the presence of MEK gene activation signature in sputum samples from COPD patients. Inhibition of MEK1/2 resulted in a superior anti-inflammatory effect in human alveolar macrophages in comparison to a p38 inhibitor. Furthermore, MEK1/2 inhibition led to an increase in bacterial killing in human neutrophils and RAW 264.7 cells that was not observed with the p38 inhibitor.Conclusion: Our data demonstrate the activation of MEK1/2 pathway in COPD and highlight a dual function of MEK1/2 inhibition in improving host defense responses whilst also controlling inflammation.Keywords: exacerbation, infection, alveolar macrophage, p38 MAPK, steroid, transcriptomics, sputum

The assessment of ADME properties and metabolic behavior of a drug is central in drug discovery and drug design. The main target for studies of metabolic properties is the Cytochrome P450 (CYP), which is responsible for the metabolism of a majority of drugs on the market and consequently also involved in many drug-drug interactions. Examples of information and tools that could guide drug design towards favorable metabolic properties are structural information of the CYPs, affinity and selectivity towards the CYPs and the site of metabolism (SOM). The present work was initiated to use experimental and computational tools to study the affinity and selectivity for two of the most important isoenzymes, CYP2D6 and CYP3A4, and highlight the benefit of combining different approaches and assays to understand the metabolic properties of a drug. A novel computational approach resulting in an assessment of enzyme-ligand interaction patterns were successfully used for a comparison of different 3D-structures in order to highlight discriminative amino acid residues. This method could also offer an identification of important interactions when understanding affinity and selectivity for an enzyme. In order to explore affinity and selectivity factors in CYP3A4 and CYP2D6 mediated metabolism two compounds were selected for an experimental evaluation of the catalytic properties of the enzyme. Based on the results from different in vitro assays it could be concluded that CYP3A4 was more unselective, producing metabolites as a result of orientations presenting many different parts of the molecules to the heme. CYP2D6 on the other hand showed more restricted binding modes. The combined information from inhibition studies and metabolite identification also gave indications on productive and non-productive binding modes in the two enzymes. With further exploration of CYP2D6, and its pharmacophore, N-dealkylation and the effect of blocking the SOM in CYP2D6 substrates were studied. These results were in agreement with the previously stated pharmacophore and also showed that the SOM for these substrates could be successfully assigned with a ligand-based approach, which could also be used to assign selectivity for CYP2D6. Key information, in designing compounds towards preferable metabolic properties, is obtained from metabolite identification or SOM determinations. I order to enhance metabolite identification a semi-automated software was tested that assigned the metabolite structure from MS raw data with high success rate. This could consequently be beneficial for drug design in that it enables a high throughput of metabolite identification data. During the course of these work important aspects to consider in drug design has been observed, e.g. improving metabolic stability by blocking soft spots tend to result in CYP inhibition. Instead of blocking soft spots the affinity for an enzyme could be diminished and this could be guided by the computational approach used in the first project. In summary, the combined knowledge from in vitro and in silico tools could be beneficial for the understanding of the metabolic behavior of a drug.