Low-cost synthesis of peptide libraries and their use for binding studies via temperature-related intensity change

Schulte, Clemens; Khayenko, Vladimir; Gupta, Amit J.; Maric, Hans Michael

doi:10.1016/j.xpro.2021.100605

Cited by 8 publications

(9 citation statements)

References 16 publications

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“…µSPOT peptide arrays 42 were synthesized using a MultiPep RSi robot (CEM GmbH, Kamp-Lindford, Germany) on in-house produced, acid-labile, amino-functionalized, cellulose membrane discs containing 9-fluorenylmethyloxycarbonyl-β-alanine (Fmoc-β-Ala) linkers (average loading: 130 nmol/disc – 4 mm diameter) 43 . Synthesis was initiated by Fmoc deprotection using 20% piperidine (pip) in dimethylformamide (DMF) followed by washing with DMF and ethanol (EtOH).…”

Section: Methodsmentioning

confidence: 99%

Multivalent binding kinetics resolved by fluorescence proximity sensing

et al. 2022

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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.

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Section: Methodsmentioning

confidence: 99%

Multivalent binding kinetics resolved by fluorescence proximity sensing

et al. 2022

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“…An MST buffer consisting of 1× phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) with 2 mM reduced L-Glutathione (GSH, Sigma Aldrich, product number G4251) and 0.05% (v/v) Tween 20 (ITW Reagents, molecular biology grade, product number A4974) was used for all MST measurements [33]. Prior to binding assays, hFSP1 WT and F360L proteins were buffer exchanged to MST buffer using 7 K MWCO ZebaTM Spin desalting columns (ThermoScientific) according to the manufacturer's instructions.…”

Section: Microscale Thermophoresis Measurementsmentioning

confidence: 99%

Molecular characterization of AIFM2/FSP1 inhibition by iFSP1-like molecules

et al. 2023

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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.

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“…In situ synthesis is also available for peptide arrays, in which the peptides are synthesised in parallel directly onto the solid surface [28]. Commercial peptide array synthesisers such as the Multipep Synthetiser (Invatis) allow for research groups to design and synthesise custom peptide arrays relevant to the autoimmune disease being studied [29].…”

Section: Peptide and Protein Fragment Arraysmentioning

confidence: 99%

Human antibody profiling technologies for autoimmune disease

2023

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Autoimmune diseases are caused by the break-down in self-tolerance mechanisms and can result in the generation of autoantibodies specific to human antigens. Human autoantigen profiling technologies such as solid surface arrays and display technologies are powerful high-throughput technologies utilised to discover and map novel autoantigens associated with disease. This review compares human autoantigen profiling technologies including the application of these approaches in chronic and post-infectious autoimmune disease. Each technology has advantages and limitations that should be considered when designing new projects to profile autoantibodies. Recent studies that have utilised these technologies across a range of diseases have highlighted marked heterogeneity in autoantibody specificity between individuals as a frequent feature. This individual heterogeneity suggests that epitope spreading maybe an important mechanism in the pathogenesis of autoimmune disease in general and likely contributes to inflammatory tissue damage and symptoms. Studies focused on identifying autoantibody biomarkers for diagnosis should use targeted data analysis to identify the rarer public epitopes and antigens, common between individuals. Thus, utilisation of human autoantigen profiling technology, combined with different analysis approaches, can illuminate both pathogenesis and biomarker discovery.

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Low-cost synthesis of peptide libraries and their use for binding studies via temperature-related intensity change

Cited by 8 publications

References 16 publications

Multivalent binding kinetics resolved by fluorescence proximity sensing

Multivalent binding kinetics resolved by fluorescence proximity sensing

Molecular characterization of AIFM2/FSP1 inhibition by iFSP1-like molecules

Human antibody profiling technologies for autoimmune disease

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