Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening

2014 ◽  
Vol 19 (7) ◽  
pp. 1000-1013 ◽  
Author(s):  
John W. Cuozzo ◽  
Holly H. Soutter
Keyword(s):  
Protein Expression ◽  
Small Molecule ◽  
Posttranslational Modifications ◽  
Expression Systems ◽  
Design Strategies ◽  
Protein Targets ◽  
Small Molecule Screening ◽  
Active Research ◽  
Small Molecule Modulators ◽  
And Function

Production of novel soluble and membrane-localized protein targets for functional and affinity-based screening has often been limited by the inability of traditional protein-expression systems to generate recombinant proteins that have properties similar to those of their endogenous counterparts. Such targets have often been labeled as challenging. Although biological validation of these challenging targets for specific disease areas may be strong, discovery of small-molecule modulators can be greatly delayed or completely halted due to target-expression issues. In this article, the limitations of traditional protein-expression systems will be discussed along with new systems designed to overcome these challenges. Recent work in this field has focused on two major areas for both soluble and membrane targets: construct-design strategies to improve expression levels and new hosts that can carry out the posttranslational modifications necessary for proper target folding and function. Another area of active research has been on the reconstitution of solubilized membrane targets for both structural analysis and screening. Finally, the potential impact of these new systems on the output of small-molecule screening campaigns will be discussed.

scholarly journals Detection of Small-Molecule Modulators of Actin-Myosin Structure and Function using High-Throughput Time-Resolved FRET

2017 ◽  
Vol 112 (3) ◽  
pp. 237a
Author(s):  
Piyali Guhathakurta ◽  
Ewa Prochniewicz ◽  
Kurt C. Peterson ◽  
Benjamin D. Grant ◽  
Gregory D. Gillispie ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Structure And Function ◽  
Throughput Time ◽  
Time Resolved ◽  
Small Molecule Modulators ◽  
And Function

scholarly journals Neuron-based high-content assay and screen for CNS active mitotherapeutics

2020 ◽  
Vol 6 (2) ◽  
pp. eaaw8702 ◽  
Author(s):  
Boglarka H. Varkuti ◽  
Miklos Kepiro ◽  
Ze Liu ◽  
Kyle Vick ◽  
Yosef Avchalumov ◽  
...  
Keyword(s):  
Small Molecule ◽  
Hippocampal Neurons ◽  
Mitochondrial Dynamics ◽  
Synaptic Activity ◽  
Glutamate Toxicity ◽  
Primary Neurons ◽  
Screening Assay ◽  
Dynamics And Function ◽  
Small Molecule Modulators ◽  
And Function

Impaired mitochondrial dynamics and function are hallmarks of many neurological and psychiatric disorders, but direct screens for mitotherapeutics using neurons have not been reported. We developed a multiplexed and high-content screening assay using primary neurons and identified 67 small-molecule modulators of neuronal mitostasis (MnMs). Most MnMs that increased mitochondrial content, length, and/or health also increased mitochondrial function without altering neurite outgrowth. A subset of MnMs protected mitochondria in primary neurons from Aβ(1–42) toxicity, glutamate toxicity, and increased oxidative stress. Some MnMs were shown to directly target mitochondria. The top MnM also increased the synaptic activity of hippocampal neurons and proved to be potent in vivo, increasing the respiration rate of brain mitochondria after administering the compound to mice. Our results offer a platform that directly queries mitostasis processes in neurons, a collection of small-molecule modulators of mitochondrial dynamics and function, and candidate molecules for mitotherapeutics.

scholarly journals AD Informer Set: Chemical tools to facilitate Alzheimer's disease drug discovery

2021 ◽  
Author(s):  
Frances M Potjewyd ◽  
Joel K Annor-Gyamfi ◽  
Jeffrey Aube ◽  
Shaoyou Chu ◽  
Ivie L Conlon ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Small Molecules ◽  
Small Molecule ◽  
Positive Control ◽  
Target Validation ◽  
Protein Targets ◽  
Novel Targets ◽  
Chemical Tools ◽  
Small Molecule Modulators

Introduction: The portfolio of novel targets to treat Alzheimer's disease (AD) has been enriched by the AMP-AD program. Methods: A cheminformatics-driven effort enabled identification of existing small molecule modulators for many protein targets nominated by AMP-AD and suitable positive control compounds to be included in the set. Results: We have built an annotated set of 171 small molecule modulators, including mostly inhibitors, targeting 98 unique proteins that have been nominated by AMP-AD consortium members as novel targets for AD treatment. These small molecules vary in their quality and should be considered chemical tools that can be used in efforts to validate therapeutic hypotheses, but which would require further optimization. A physical copy of the AD Informer Set can be ordered via the AD Knowledge Portal. Discussion: Small molecule tools that enable target validation are important tools for the translation of novel hypotheses into viable therapeutic strategies for AD.

scholarly journals Turning the tide on Alzheimer’s disease: modulation of γ-secretase

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Joanna E. Luo ◽  
Yue-Ming Li
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Neurodegenerative Disorder ◽  
Complex Structure ◽  
Aβ Peptides ◽  
Small Molecule Probes ◽  
Small Molecule Modulators ◽  
And Function

AbstractAlzheimer’s disease (AD) is the most common type of neurodegenerative disorder. Amyloid-beta (Aβ) plaques are integral to the “amyloid hypothesis,” which states that the accumulation of Aβ peptides triggers a cascade of pathological events leading to neurodegeneration and ultimately AD. While the FDA approved aducanumab, the first Aβ-targeted therapy, multiple safe and effective treatments will be needed to target the complex pathologies of AD. γ-Secretase is an intramembrane aspartyl protease that is critical for the generation of Aβ peptides. Activity and specificity of γ-secretase are regulated by both obligatory subunits and modulatory proteins. Due to its complex structure and function and early clinical failures with pan inhibitors, γ-secretase has been a challenging drug target for AD. γ-secretase modulators, however, have dramatically shifted the approach to targeting γ-secretase. Here we review γ-secretase and small molecule modulators, from the initial characterization of a subset of NSAIDs to the most recent clinical candidates. We also discuss the chemical biology of γ-secretase, in which small molecule probes enabled structural and functional insights into γ-secretase before the emergence of high-resolution structural studies. Finally, we discuss the recent crystal structures of γ-secretase, which have provided valuable perspectives on substrate recognition and molecular mechanisms of small molecules. We conclude that modulation of γ-secretase will be part of a new wave of AD therapeutics.

Let there be light: how to use photoswitchable cross-linker to reprogram proteins

2017 ◽  
Vol 45 (3) ◽  
pp. 831-837 ◽  
Author(s):  
Daniel Hoersch
Keyword(s):  
Rational Design ◽  
Design Strategies ◽  
Molecular Tweezers ◽  
Protein Targets ◽  
Reactive Groups ◽  
Synthetic Cells ◽  
End Distance ◽  
Cross Linker ◽  
Cellular Pathways ◽  
And Function

Azobenzene is a photo-isomerizing molecule whose end-to-end distance changes upon external illumination. When combined with site-specific reactive groups, it can be used as molecular tweezers to remote-control the structure and function of protein targets. The present study gives a brief overview over the rational design strategies that use an azobenzene-based photoswitchable cross-linker to engineer ON/OFF switches into functional proteins or to reprogram proteins for novel functions. The re-engineered proteins may be used as remote controls for cellular pathways, as light-gated drug delivery platforms or as light-powered machinery of synthetic cells and micro-scaled factories.

P3.12 Small molecule compounds that correct alternative splicing of the SMN2 gene and restore SMN protein expression and function

2011 ◽  
Vol 21 (9-10) ◽  
pp. 685
Author(s):  
N.A. Naryshkin ◽  
J. Narasimhan ◽  
A. Dakka ◽  
V. Gabbeta ◽  
M. Haley ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Protein Expression ◽  
Small Molecule ◽  
Correct Alternative ◽  
Smn2 Gene ◽  
Smn Protein ◽  
And Function

scholarly journals N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

2021 ◽  
Vol 12 ◽  
Author(s):  
Yun-Ji Shin ◽  
Julia König-Beihammer ◽  
Ulrike Vavra ◽  
Jennifer Schwestka ◽  
Nikolaus F. Kienzl ◽  
...  
Keyword(s):  
Protein Expression ◽  
Recombinant Proteins ◽  
Receptor Binding ◽  
Posttranslational Modifications ◽  
Functional Expression ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Receptor Binding Domain ◽  
And Function ◽  
The Impact

Nicotiana benthamiana is used worldwide as production host for recombinant proteins. Many recombinant proteins such as monoclonal antibodies, growth factors or viral antigens require posttranslational modifications like glycosylation for their function. Here, we transiently expressed different variants of the glycosylated receptor binding domain (RBD) from the SARS-CoV-2 spike protein in N. benthamiana. We characterized the impact of variations in RBD-length and posttranslational modifications on protein expression, yield and functionality. We found that a truncated RBD variant (RBD-215) consisting of amino acids Arg319-Leu533 can be efficiently expressed as a secreted soluble protein. Purified RBD-215 was mainly present as a monomer and showed binding to the conformation-dependent antibody CR3022, the cellular receptor angiotensin converting enzyme 2 (ACE2) and to antibodies present in convalescent sera. Expression of RBD-215 in glycoengineered ΔXT/FT plants resulted in the generation of complex N-glycans on both N-glycosylation sites. While site-directed mutagenesis showed that the N-glycans are important for proper RBD folding, differences in N-glycan processing had no effect on protein expression and function.

scholarly journals Development of Homogeneous Nonradioactive Methyltransferase and Demethylase Assays Targeting Histone H3 Lysine 4

2011 ◽  
Vol 17 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Nancy Gauthier ◽  
Mireille Caron ◽  
Liliana Pedro ◽  
Mathieu Arcand ◽  
Julie Blouin ◽  
...  
Keyword(s):  
Small Molecule ◽  
Histone Methylation ◽  
Posttranslational Modifications ◽  
Histone H3 ◽  
Biochemical Screening ◽  
Histone Methyltransferases ◽  
N Terminus ◽  
Cancer Inflammation ◽  
Small Molecule Modulators ◽  
Significant Attention

Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z′ factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.

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