Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is ...tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone-reader interactions, but not the transient interactions of Zn
-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.
Ferroptosis is a form of cell death characterized by phospholipid peroxidation, where numerous studies have suggested that the induction of ferroptosis is a therapeutic strategy to target therapy ...refractory cancer entities. Ferroptosis suppressor protein 1 (FSP1), an NAD(P)H-ubiquinone reductase, is a key determinant of ferroptosis vulnerability, and its pharmacological inhibition was shown to strongly sensitize cancer cells to ferroptosis. A first generation of FSP1 inhibitors, exemplified by the small molecule iFSP1, has been reported; however, the molecular mechanisms underlying inhibition have not been characterized in detail. In this study, we explore the species-specific inhibition of iFSP1 on the human isoform to gain insights into its mechanism of action. Using a combination of cellular, biochemical, and computational methods, we establish a critical contribution of a species-specific aromatic architecture that is essential for target engagement. The results described here provide valuable insights for the rational development of second-generation FSP1 inhibitors combined with a tracer for screening the druggable pocket. In addition, we pose a cautionary notice for using iFSP1 in animal models, specifically murine models.
The Hippo signalling pathway and its central effector YAP regulate proliferation of cardiomyocytes and growth of the heart. Using genetic models in mice we show that the increased proliferation of ...embryonal and postnatal cardiomyocytes due to loss of the Hippo-signaling component SAV1 depends on the Myb-MuvB (MMB) complex. Similarly, proliferation of postnatal cardiomyocytes induced by constitutive active YAP requires MMB. Genome studies revealed that YAP and MMB regulate an overlapping set of cell cycle genes in cardiomyocytes. Protein-protein interaction studies in cell lines and with recombinant proteins showed that YAP binds directly to B-MYB, a subunit of MMB, in a manner dependent on the YAP WW domains and a PPXY motif in B-MYB. Disruption of the interaction by overexpression of the YAP binding domain of B-MYB strongly inhibits the proliferation of cardiomyocytes. Our results point to MMB as a critical downstream effector of YAP in the control of cardiomyocyte proliferation.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Protein-peptide interactions are involved in many fundamental cellular functions and constitute promising drug targets. Here, we provide a detailed protocol for the cost-effective preparation of a ...cellulose-based solid support for synthesis of nanoscale to micromolar-scale peptide libraries. Their subsequent use for high-throughput protein interaction screening as well as affinity determination in solution provides binding data for thousands of unique peptides with a turnover of 1 to 2 weeks, thereby facilitating in vitro assessment and development of high-affinity binders.
For complete details on the use and execution of this protocol, please refer to Schulte et al., (2020)
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•Facile and cost-effective preparation of a cellulose-based solid support•Automated peptide synthesis on cellulose disks after laser perforation•Seamless transition from peptide synthesis to the binding assay•Quasi-label-free protein affinity determination in solution
Protein-peptide interactions are involved in many fundamental cellular functions and constitute promising drug targets. Here, we provide a detailed protocol for the cost-effective preparation of a cellulose-based solid support for synthesis of nanoscale to micromolar-scale peptide libraries. Their subsequent use for high-throughput protein interaction screening as well as affinity determination in solution provides binding data for thousands of unique peptides with a turnover of 1 to 2 weeks, thereby facilitating in vitro assessment and development of high-affinity binders.
Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological ...recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.
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•Protein affinity determination via temperature-related intensity change (TRIC)•Tracer displacement enables sensitive and quasi-label-free measurements in solution•Coupling to high-throughput peptide synthesis empowers large-scaled screening•Largely automated hit identification and affinity determination in nanomolar scale
Biochemistry Methods, Neuroscience, Biophysical Methods, Biochemistry, Biophysics;
Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies ...and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.
Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, ...assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.
Drugs directly targeting γ-aminobutyric acid type A receptors (GABAARs), the major mediators of fast synaptic inhibition, contribute significantly to today's neuropharmacology. Emerging evidence ...establishes intracellularly GABAAR-associated proteins as the central players in determining cellular and subcellular GABAergic input sites, thereby providing pharmacological opportunities to affect distinct receptor populations and address discrete neuronal dysfunctions. Here, we report on recently studied GABAAR-associated proteins and highlight challenges and newly available methods for their functional and physical mapping. We anticipate these efforts to contribute to decipher the complexity of GABAergic signalling in the brain and eventually enable therapeutic avenues for, so far, untreatable neuronal disorders and diseases.
•Associated proteins determine GABAAR distribution, function, and pharmacology.•Their targeting enables circuit-specific manipulation of GABAergic signalling.•New methods enable the definition and targeting of the receptor-associated proteins.
Drugs directly targeting γ-aminobutyric acid type A receptors (GABA
Rs), the major mediators of fast synaptic inhibition, contribute significantly to today's neuropharmacology. Emerging evidence ...establishes intracellularly GABA
R-associated proteins as the central players in determining cellular and subcellular GABAergic input sites, thereby providing pharmacological opportunities to affect distinct receptor populations and address discrete neuronal dysfunctions. Here, we report on recently studied GABA
R-associated proteins and highlight challenges and newly available methods for their functional and physical mapping. We anticipate these efforts to contribute to decipher the complexity of GABAergic signalling in the brain and eventually enable therapeutic avenues for, so far, untreatable neuronal disorders and diseases.
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Background: Platinum-based chemotherapy (PBC) is the standard-of-care first-line (1L) tx for pts with la/mUC (followed by avelumab 1L maintenance in pts without progressive disease since its ...approval in Germany in Jan 2021). This study describes demographics, tx patterns, and clinical outcomes for pts with la/mUC treated in routine clinical practice in Germany between 2019 and 2021. Methods: CONVINCE, a retrospective, multicenter medical chart review study, was initiated in Dec 2021 and included adult pts who received 1L tx for la/mUC between 2019 and 2021. All pts were required to have ≥6 months of follow-up data available after end/start of 1L, depending on the type of tx received. Fully anonymized data were obtained from 27 oncology or urology institutions (8 hospitals and 19 office-based practices) across Germany. Descriptive statistics were used to analyze the results, and Kaplan-Meier methods were used to compute time-to-event outcomes. Pts were classified into 3 groups: (A) PBC with end of tx between Jan 2019 and Sep 2021 and (B) immuno-oncology (IO) therapy and (C) other non-PBC tx, both with start of tx between Jan 2019 and Sep 2021. Results: Of 188 pts included, median age at start of 1L was 70 years, 72.3% were male, most had ECOG PS 0/1 (92.5%), and the majority (60.1%) were treated by medical oncologists. A total of 143 pts (group A) received PBC (PBC + gemcitabine, 88.1%; PBC + non-gemcitabine agents, 8.4%; and cisplatin monotherapy, 3.5%). Cisplatin + gemcitabine (82.5%) was used more often than carboplatin + gemcitabine (17.5%). Six pts were treated with avelumab 1L maintenance following PBC. In group B, 36 pts (19.1%) received IO monotherapy (atezolizumab, 44.4%; pembrolizumab, 41.7%). In group C, 9 pts (4.8%) received non-PBC tx. Clinical outcomes in groups A and B are shown in the Table. Results for group C are not shown due to small sample size. Conclusions: CONVINCE describes characteristics, tx patterns, and outcomes in pts with la/mUC in routine clinical practice in Germany. In adherence with tx guidelines, most pts received PBC in 1L. New IO approvals, including avelumab 1L maintenance and other novel agents, could improve outcomes in pts with la/mUC. Further real-world studies should be performed to evaluate the uptake of these options within routine care. Table: see text
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