Institutes
Refine
Language
- English (25)
Has Fulltext
- yes (25)
Is part of the Bibliography
- no (25)
Keywords
- Biochemistry (2)
- Biophysical chemistry (2)
- PROTAC (2)
- Structural biology (2)
- ATP binding (1)
- Apoptosis (1)
- Aurora kinase (1)
- BET proteins (1)
- BH3 mimetics (1)
- BioID (1)
Phenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still pose a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies.
Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.
A toolbox for the generation of chemical probes for Baculovirus IAP Repeat containing proteins
(2022)
E3 ligases constitute a large and diverse family of proteins that play a central role in regulating protein homeostasis by recruiting substrate proteins via recruitment domains to the proteasomal degradation machinery. Small molecules can either inhibit, modulate or hijack E3 function. The latter class of small molecules led to the development of selective protein degraders, such as PROTACs (PROteolysis TArgeting Chimeras), that recruit protein targets to the ubiquitin system leading to a new class of pharmacologically active drugs and to new therapeutic options. Recent efforts have focused on the E3 family of Baculovirus IAP Repeat (BIR) domains that comprise a structurally conserved but diverse 70 amino acid long protein interaction domain. In the human proteome, 16 BIR domains have been identified, among them promising drug targets such as the Inhibitors of Apoptosis (IAP) family, that typically contain three BIR domains (BIR1, BIR2, and BIR3). To date, this target area lacks assay tools that would allow comprehensive evaluation of inhibitor selectivity. As a consequence, the selectivity of current BIR domain targeting inhibitors is unknown. To this end, we developed assays that allow determination of inhibitor selectivity in vitro as well as in cellulo. Using this toolbox, we have characterized available BIR domain inhibitors. The characterized chemical starting points and selectivity data will be the basis for the generation of new chemical probes for IAP proteins with well-characterized mode of action and provide the basis for future drug discovery efforts and the development of PROTACs and molecular glues.
Targeted protein degradation (TPD) has recently emerged as an exciting new drug modality. However, the strategy of developing small molecule-based protein degraders has evolved over the past two decades and has now established molecular tags that are already in clinical use, as well as chimeric molecules, PROteolysis TArgeting Chimeras (PROTACs), based mainly on ligand systems developed for the two E3 ligases CRBN and VHL. The large size of the human E3 ligase family suggests that PROTACs can be developed by targeting a large diversity of E3 ligases, some of which have restricted expression patterns with the potential to design disease- or tissue-specific degraders. Indeed, many new E3 ligands have been published recently, confirming the druggability of E3 ligases. This review summarises recent data on E3 ligases and highlights the challenges in developing these molecules into efficient PROTACs rivalling the established degrader systems.
Isothermal titration calorimetry (ITC) is a widely used technique for the characterization of protein-protein and protein-ligand interactions. It provides information on the stoichiometry, affinity, and the thermodynamic driving forces of interactions. This chapter exemplifies the use of ITC to investigate interactions between human autophagy modifiers (LC3/GABARAP proteins) and their interaction partners, the LIR motif containing sequences. The purpose of this report is to present a detailed protocol for the production of LC3/GABARAP-interacting LIR peptides using E. coli expression systems. In addition, we outline the design of ITC experiments using the LC3/GABARAP:peptide interactions as an example. Comprehensive troubleshooting notes are provided to facilitate the adaptation of these protocols to different ligand-receptor systems. The methodology outlined for studying protein-ligand interactions will help to avoid common errors and misinterpretations of experimental results.