scholarly journals Modulating protein activity using tethered ligands with mutually exclusive binding sites

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Alberto Schena ◽  
Rudolf Griss ◽  
Kai Johnsson
Keyword(s):  
Small Molecules ◽  
Binding Sites ◽  
Protein A ◽  
Living Cells ◽  
Protein Activity ◽  
Research Areas ◽  
Specific Effector ◽  
Tethered Ligands ◽  
General Method

Abstract The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand–protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors.

scholarly journals Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

2017 ◽  
Vol 114 (4) ◽  
pp. 681-686 ◽  
Author(s):  
Glen P. Liszczak ◽  
Zachary Z. Brown ◽  
Samuel H. Kim ◽  
Rob C. Oslund ◽  
Yael David ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chemical Synthesis ◽  
Small Molecules ◽  
Small Molecule ◽  
Binding Sites ◽  
Dna Binding Protein ◽  
Dna Binding Proteins ◽  
Live Cells ◽  
Binding Partners

Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

scholarly journals Binding of Gal4p and Bicoid to Nucleosomal Sites in Yeast in the Absence of Replication

1999 ◽  
Vol 19 (4) ◽  
pp. 2977-2985 ◽  
Author(s):  
Bhuvana Balasubramanian ◽  
Randall H. Morse
Keyword(s):  
Transcription Factors ◽  
Dna Replication ◽  
Binding Sites ◽  
Transcriptional Activator ◽  
Nucleosome Positioning ◽  
Living Cells ◽  
Intracellular Environment ◽  
Nucleosomal Dna

ABSTRACT The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it is unable to access in vitro. One event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites in yeast, we have used previously characterized chromatin reporters in which Gal4p binding sites are incorporated into nucleosomes. We find that Gal4p is able to perturb nucleosome positioning via nucleosomal binding sites in yeast arrested either in G1, with α-factor, or in G2/M, with nocodazole. Similar results were obtained whether Gal4p synthesis was induced from the endogenous promoter by growth in galactose medium or by an artificial, hormone-inducible system. We also examined binding of theDrosophila transcriptional activator Bicoid, which belongs to the homeodomain class of transcription factors. We show that Bicoid, like Gal4p, can bind to nucleosomal sites inSWI + and swi1Δ yeast and in the absence of replication. Our results indicate that some feature of the intracellular environment other than DNA replication or the SWI-SNF complex permits factor access to nucleosomal sites.

scholarly journals Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes

2005 ◽  
Vol 280 (16) ◽  
pp. 15553-15560 ◽  
Author(s):  
Rajesh Mishra ◽  
Robert Seckler ◽  
Rajiv Bhat
Keyword(s):  
Small Molecules ◽  
Molecular Chaperones ◽  
Rational Design ◽  
Protein Production ◽  
Citrate Synthase ◽  
Protein A ◽  
Complete Recovery ◽  
Hydroxyl Groups ◽  
Native State

Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7mglycerol and 10 μg/ml protein, a result superior to the action of the molecular chaperones GroEL and GroESin vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.

Structure-based virtual screening towards the discovery of novel FOXM1 inhibitors

2021 ◽  
Author(s):  
Zhuo-Song Xie ◽  
Zi-Ying Zhou ◽  
Lian-Qi Sun ◽  
Hong Yi ◽  
Si-Tu Xue ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Virtual Screening ◽  
Mouse Model ◽  
Small Molecules ◽  
Antiproliferative Activity ◽  
Binding Sites ◽  
Specific Inhibitor ◽  
Activity Analysis ◽  
Computer Aided Drug Design

Aim: Given the importance of FOXM1 in the treatment of ovarian cancer, we aimed to identify an excellent specific inhibitor and examined its underlying therapeutic effect. Materials & methods: The binding statistics for FDI-6 with FOXM1 were calculated through computer-aided drug design (CADD). We selected XST-119 through virtual screening, performed surface plasmon resonance and in vitro cell antiproliferative activity analysis and evaluated its antitumor efficacy in a mouse model. Results: XST-119 had significantly higher affinity for FOXM1 and antiproliferative activity than FDI-6. XST-119 had a definite inhibitory activity in a xenograft mouse model. Conclusion: We identified XST-119, a FOXM1 inhibitor, with better efficacy for treatment of ovarian cancer. FOXM1 binding sites for small molecules are also highlighted, which may provide the foundation for further drug discovery.

scholarly journals A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brent Townshend ◽  
Joy S. Xiang ◽  
Gabriel Manzanarez ◽  
Eric J. Hayden ◽  
Christina D. Smolke
Keyword(s):  
Small Molecules ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Living Cells ◽  
Regulate Gene Expression ◽  
Liquid Handling ◽  
Regulate Gene

AbstractBiosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

scholarly journals A Bioluminescent Biosensor for Quantifying the Interaction of SARS-CoV-2 and Its Receptor ACE2 in Cells and In Vitro

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1055
Author(s):  
Xiaolong Yang ◽  
Lidong Liu ◽  
Yawei Hao ◽  
Eva So ◽  
Sahar Sarmasti Emami ◽  
...  
Keyword(s):  
Small Molecules ◽  
Living Cells ◽  
Cellular Receptor ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Discovery Process ◽  
Drug Discovery Process ◽  
Angiotensin Converting Enzyme 2 ◽  
Host Interactions

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is currently spreading and mutating with increasing speed worldwide. Therefore, there is an urgent need for a simple, sensitive, and high-throughput (HTP) assay to quantify virus–host interactions in order to quickly evaluate the infectious ability of mutant viruses and to develop or validate virus-inhibiting drugs. Here, we developed an ultrasensitive bioluminescent biosensor to evaluate virus–cell interactions by quantifying the interaction between the SARS-CoV-2 receptor binding domain (RBD) and its cellular receptor angiotensin-converting enzyme 2 (ACE2) both in living cells and in vitro. We have successfully used this novel biosensor to analyze SARS-CoV-2 RBD mutants and evaluated candidate small molecules (SMs), antibodies, and peptides that may block RBD:ACE2 interaction. This simple, rapid, and HTP biosensor tool will significantly expedite the detection of viral mutants and the anti-COVID-19 drug discovery process.

scholarly journals A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2

2021 ◽  
Vol 7 (15) ◽  
pp. eabg1950
Author(s):  
Nicolas Bery ◽  
Carole J.R. Bataille ◽  
Angela Russell ◽  
Angela Hayes ◽  
Florence Raynaud ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
Binding Sites ◽  
Screening Method ◽  
Chromosomal Translocation ◽  
Resonance Method ◽  
Protein Protein Interactions ◽  
Intracellular Antibody ◽  
A Cell

Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody–guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.

scholarly journals A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

2020 ◽  
Author(s):  
Brent Townshend ◽  
Joy Xiang ◽  
Gabriel Manzanarez ◽  
Eric Hayden ◽  
Christina Smolke
Keyword(s):  
Small Molecules ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Living Cells ◽  
Regulate Gene Expression ◽  
Liquid Handling ◽  
Regulate Gene

AbstractBiosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Using aptamer-coupled ribozyme libraries and a novel ribozyme regeneration method, we developed de novo rapid in vitro evolution of RNA biosensors (DRIVER) that enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identified and validated biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors were applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors were also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

scholarly journals Necator americanus Ancylostoma Secreted Protein-2 (Na-ASP-2) Binds an Ascaroside (ascr#3) in Its Fatty Acid Binding Site

2020 ◽  
Vol 8 ◽  
Author(s):  
Ola El Atab ◽  
Rabih Darwiche ◽  
Nathanyal J. Truax ◽  
Roger Schneiter ◽  
Kenneth G. Hull ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Small Molecules ◽  
Binding Sites ◽  
Structural Basis ◽  
Side Chain ◽  
Secreted Protein ◽  
Competition Assay ◽  
Fatty Acid Binding ◽  
Binding Cavity

During their infective stages, hookworms release excretory-secretory (E-S) products, small molecules, and proteins to help evade and suppress the host's immune system. Small molecules found in E-S products of mammalian hookworms include nematode derived metabolites like ascarosides, which are composed of the sugar ascarylose linked to a fatty acid side chain. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). In this study, two ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using both a ligand competition assay and microscale thermophoresis. The fatty acid moieties of both ascarosides tested and ascr#3, an ascaroside found in rat hookworm E-S products, bind to Na-ASP-2's palmitate binding cavity. These molecules were confirmed to bind to the palmitate but not the sterol binding sites. An ascaroside, oscr#10, which is not found in hookworm E-S products, does not bind to Na-ASP-2. More studies are required to determine the structural basis of ascarosides binding by Na-ASP-2 and to understand the physiological significance of these observations.

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