Refolding of Escherichia coli produced membrane protein inclusion bodies immobilised by nickel chelating chromatography

Two distinctly different membrane proteins, which produced inclusion bodies in Escherichia coli, have been refolded to reconstitute properties appropriate to their native counterparts. The method employed utilises nickel chelating chromatography, where the solubilised inclusion bodies bind, refold and elute. Our aims were to release a large pool of membrane protein for functional, mutational and crystallisation screening studies. It is hoped that the methods described here will have a general application for other membrane proteins which have formed inclusion bodies

Thresholds of toxicological concern - overview of ongoing scientific developments

The Concept of the Threshold of Toxicological Concern (TTC) uses the principles of chemical grouping and read-across to screen chemicals at low levels of exposure for prioritization of follow-up testing. The approach incorporates consideration of chemical structure, metabolism, and animal toxicity data to establish chronic exposure thresholds below which there is no appreciable human health risk. The chemical groups and exposure thresholds were originally established based on the analysis by Rulis, Cramer and Munro. The TTC concept has recently been re-evaluated by several expert working groups and acknowledged for some applications by a number of regulatory bodies such as EFSA and JECFA. However, controversy still surrounds the use of the approach due to actual or perceived concerns such as imprecise structural groupings, old or non-validated datasets, impractically low thresholds, the use of 5th percentiles as cut-off. At the same time, safety and risk assessment scientists are striving to identify methods and approaches capable of achieving the vision to Refine, Replace and Reduce animal testing, whilst trying to improve the efficiency and throughput of low tier evaluations. Consequently, several activities have been initiated or performed by different research groups to update the TTC datasets with more, recent and structured data including improved study documentation and dose selection information. Activities on the different levels aiming to expand the chemical domain of the TTC concept and to refine the structural grouping applied will be presented and future research needs discussed

Treating Cancer by Spindle Assembly Checkpoint Abrogation Discovery of Two Clinical Candidates, BAY 1161909 and BAY 1217389, Targeting MPS1 Kinase

Inhibition of monopolar spindle 1 MPS1 kinase represents a novel approach to cancer treatment instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series triazolopyridines and imidazopyrazines . The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10 fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy model

Treating Cancer by Spindle Assembly Checkpoint Abrogation: Discovery of Two Clinical Candidates, BAY 1161909 and BAY 1217389, Targeting MPS1 Kinase

Inhibition of monopolar spindle 1 MPS1 kinase represents a novel approach to cancer treatment instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series triazolopyridines and imidazopyrazines . The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10 fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy model