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Modulation of HCA by tacrine on living cells. (a) Cartoon of CLASH-AChE/HCA. The acetylcholine esterase (AChE) inhibitor tacrine (T) displaces edrophonium (E) from AChE and benzenesulfonamide (SA) can bind to HCA inducing an increase in FRET efficiency. (b) Structure of the labelling compound. (c) Fluorescence micrographs of HEK293 cells displaying CLASH-AChE/HCA on their surface. In the absence of tacrine Cy3 emission is more intense, while on addition tacrine Cy5 emission increases; scale bars, 50 μm. (d) Changes observed in FRET ratio, donor (Cy3) and acceptor (Cy5) emission on perfusion of HEK293 cells displaying CLASH-AChE/HCA on their surface with 3 mM tacrine.
Source publication
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 approac...
Contexts in source publication
Context 1
... create CLASH-AChE/HCA, AChE was fused to the N-terminus of an HCA-based SNIFIT (Fig. 4a). We targeted the construct to the outer cell membrane of transiently transfected HEK293 cells. The cells were labelled with a dual ligand containing benzenesulfonamide as primary ligand and edrophonium, an inhibitor of AChE 26 , as secondary ligand (Fig. 4b). In the absence of free AChE inhibitors, the stronger interaction of ...
Context 2
... create CLASH-AChE/HCA, AChE was fused to the N-terminus of an HCA-based SNIFIT (Fig. 4a). We targeted the construct to the outer cell membrane of transiently transfected HEK293 cells. The cells were labelled with a dual ligand containing benzenesulfonamide as primary ligand and edrophonium, an inhibitor of AChE 26 , as secondary ligand (Fig. 4b). In the absence of free AChE inhibitors, the stronger interaction of edrophonium with AChE prevents the binding of the primary ligand to HCA, keeping the sensor in its low-FRET state. When the AChE inhibitor tacrine, a drug used in the past to treat Alzheimer's disease, was perfused on living cells expressing CLASH-AChE/HCA, tacrine ...
Context 3
... ligand to HCA, keeping the sensor in its low-FRET state. When the AChE inhibitor tacrine, a drug used in the past to treat Alzheimer's disease, was perfused on living cells expressing CLASH-AChE/HCA, tacrine displaced the secondary ligand from AChE, allowing the benzenesulfonamide to bind HCA and leading to high FRET efficiency of the sensor (Fig. 4c-d). Importantly, the switching between the two states was fully reversible, and the overall measured FRET ratio change was twofold higher than the original ACh-SNIFIT 26 . In CLASH- AChE/HCA, the relative strength of the two synthetic ligands is such that HCA is active in the absence of the effector targeting AChE and inhibited by its ...
Citations
... "Vesicular glue" for molecular recognition of proteins Modulation of the enzymatic activity of proteins by synthetic materials is an important aspect of protein-based bioengineering as well as to understand the diverse range of reactions inside the cells and predict the metabolism of living things. 73 Therefore, we investigated the possibility of temporally regulating the activity of an enzyme using the self-assembled C 16 -VesiGlue. Given the importance of CytC enzyme in the electron transport chain, 74 we studied the peroxidase activity of CytC templated on the vesicle surfaces. ...
Regulation of enzyme activity and biocatalytic cascades on compartmentalized cellular components is key to the adaptation of cellular processes such as signal transduction and metabolism in response to varying external conditions. Synthetic molecular glues have enabled enzyme inhibition and regulation of protein-protein interactions. So far, all the molecular glue systems based on covalent interactions operated under steady state conditions. To emulate dynamic biological processes under dissipative conditions, we introduce herein a transient supramolecular glue with a controllable lifetime. The transient system uses multivalent supramolecular interactions between guanidinium group-bearing surfactants and adenosine triphosphates (ATP), resulting in bilayer vesicle structures. Unlike the conventional chemical agents for dissipative assemblies, ATP here plays the dual role of providing a structural component for the assembly as well as presenting active functional groups to “glue” enzymes on the surface. While glueing of the enzymes on the vesicles achieves augmented catalysis, oscillation of ATP concentration allows temporal control of the catalytic activities similar to the dissipative cellular nanoreactors. We further demonstrate temporal upregulation and control of complex biocatalytic reaction networks on the vesicles. Altogether, the temporal activation of biocatalytic cascades on the dissipative vesicular glue presents an adaptable and dynamic system emulating heterogeneous cellular processes, opening up avenues for effective protocell construction and therapeutic interventions.
... Since the two encoded handles behave like a ''blank slate,'' one can envision dual labeling systems using virtually any functionality. Included among them is the attachment of two distinct prosthetic groups, such as stimuli-responsive polymers, 176,177 tethered small-molecule regulators, 39,178 and/or multi-modal probes to generate multi-functional protein-based biosensors with func-tionalities that cannot be accessed naturally or with current technologies. Using this DEAL approach, we envision that virtually any protein can become a chassis for extended biochemical functionality for both in vivo and in vitro applications. ...
The ability to selectively modify proteins at two or more defined locations opens new avenues for manipulating, engineering, and studying living systems. As a chemical biology tool for the site-specific encoding of non-canonical amino acids into proteins in vivo, genetic code expansion (GCE) represents a powerful tool to achieve such modifications with minimal disruption to structure and function through a two-step "dual encoding and labeling" (DEAL) process. In this review, we summarize the state of the field of DEAL using GCE. In doing so, we describe the basic principles of GCE-based DEAL, catalog compatible encoding systems and reactions, explore demonstrated and potential applications, highlight emerging paradigms in DEAL methodologies, and propose novel solutions to current limitations.
... The probe was shown to conjugate to a SNAP-tag fusion protein and then with either luciferase or streptavidin but, due to steric hindrance, not both. 62 Another multifunctional probe was recently introduced for photoproximity protein interaction (PhotoPPI) profiling in live cells. 63 Protein aggregation is associated with several neurodegenerative diseases and many unanswered questions relating to protein aggregation remain due to a lack of tools for visualization. ...
The conjugation of proteins with synthetic molecules can be conducted in many different ways. In this Perspective, we focus on tag-based techniques and specifically on the SNAP-tag technology. The SNAP-tag technology makes use of a fusion protein between a protein of interest and an enzyme tag that enables the actual conjugation reaction. The SNAP-tag is based on the O6-alkylguanine-DNA alkyltransferase (AGT) enzyme and is optimized to react selectively with O6-benzylguanine (BG) substrates. BG-containing dye derivatives have frequently been used to introduce a fluorescent tag to a specific protein. We believe that the site-specific conjugation of polymers to proteins can significantly benefit from the SNAP-tag technology. Especially, polymers synthesized via reversible deactivation radical polymerization allow for the facile introduction of a BG end group to enable SNAP-tag conjugation.
... For docking studies, all the selected OS bioactive compounds were docked against filarial target proteins GST, Trx, GR, and SOD. PatchDock server (parameter RMSD value 1.5, protein complex-small ligand) (Schneidman-Duhovny et al. 2005) and YASARA (Yet Another Scientific Artificial Reality Application) (Krieger and Vriend 2014) tools were used for docking of ligands with target molecules. The best docking configuration of OS bioactive compounds and filarial antioxidant proteins/enzymes was visualized with Biovia Discovery studio 3.5 (Kumar et al. 2022). ...
... The maximum interactions of Ocimum bioactive compounds with filarial antioxidant proteins were present within the active site of GST, Trx, GR, and SOD ( Figure 5). It is an established fact that binding of ligands within the active site of target molecules can lead to modulations in enzymatic functions and molecular properties of proteins (Schena et al. 2015). We have also analyzed the H-bonding between OS bioactive compounds and target proteins and the maximum number of H-bonds were formed between GST and rosmarinic acid. ...
Context
The anthelminthic effect of Ocimum species (Lamiaceae) has been reported, however, its anti-filarial effect has not been explored to date.
Objective
This study evaluates the effect of Ocimum sanctum L. (OS) against lymphatic filarial parasites.
Material and methods
The ethanol extract of OS (EOS) leaves was tested for anti-filarial activity against Setaria cervi. Equal size and number (n = 10) of adult female S. cervi worms were incubated in 125, 250 or 375 μg/mL EOS extract for 6 h at 37 °C. The OS bioactive components were identified by UPLC-ESI-MS/MS and subjected to docking and molecular dynamics (MD) simulation against filarial antioxidant proteins.
Results
The EOS significantly inhibited the motility of adult female S. cervi after 6 h of incubation. The motility was found to be reduced by 53.7% in 375 µg/mL and 43.8% in 250 µg/mL EOS after 6 h of treatment. The UPLC-ESI-MS/MS analysis of ethanol extract of O. sanctum revealed the presence of 13 bioactive compounds. The docking analysis showed eight OS bioactive compounds to have high binding affinity (> 4.8 kcal/mol) towards antioxidant proteins of filarial parasites. Additionally, MD simulation studies showed significant impact of (RMSD ≤ 10 Å) chlorogenic acid, luteolin and ursolic acid on filarial antioxidant enzymes/proteins. To our knowledge, this is the first report of the anti-filarial activity of Ocimum sanctum.
Discussion and conclusions
The effect of EOS and OS bioactive components on human filarial parasites can be further evaluated for the development of new anti-filarial formulations.
... Modulation of the enzymatic activity of proteins by synthetic materials is an important aspect in protein-based bioengineering as well as to understand the diverse range of reactions inside the cells and predict the metabolism of living things. 64 Therefore, we investigated the possibility of temporally regulating the activity of an enzyme using the self-assembled C16VesiGlue. Given the importance of CytC enzyme in the electron transport chain, 65 we studied the peroxidase activity of CytC templated on the vesicle surfaces. ...
Regulation of enzyme activity is key to the adaptation of cellular processes such as signal transduction and metabolism in response to varying external conditions. Synthetic molecular glues have provided effective systems for enzyme inhibition and regulation of protein-protein interactions. So far, all the molecular glue systems based on covalent interactions operated in equilibrium conditions. To emulate dynamic far-from-equilibrium biological processes, we introduce herein a transient supramolecular glue with controllable lifetime. The transient system uses multivalent supramolecular interactions between guanidium group-bearing surfactants and adenosine triphosphates (ATP), resulting in bilayer vesicle structures. Unlike the conventional fuels for non-equilibrium assemblies, ATP here plays the dual role of providing a structural component for the assembly as well as presenting active functional groups to “glue” enzymes on the surface.
... The targets used for docking analysis with 9H-thioxanthene drugs 1-4 are VEGFR-2 and COX-2, which have been extensively revealed to contribute to apoptotic regulation, cell-cycle progression, transcriptional regulation, DNA damage repair, stem-cell self-renewal, metabolism, spermatogenesis, and neuronal function and antiangiogenesis [19,20,22,23,[49][50][51][52][53][54]. The ligand binding in the active site of a target is suggestive of the possibility that the ligand may be capable of driving functional alteration of the target molecules [55,56]. Drug-target interactions were also deciphered in terms of interacting amino acid residues, hydrogen bonding, docking energy analysis, and comparisons of active site amino acid residues and probable binding sites. ...
The approach of using existing drugs initially developed for one disease to treat other indications has found success across medical fields. This article emphasizes the drug repurposing of 9H-thioxanthene based on FDA-approved drugs for anticancer agents precisely targeting VEGFR-2 and COX-2. The investigated 9H-thioxanthene drugs 1-4 were analyzed for Lipinski's drug-likeness rule and ideal ADME parameters. The results show that all calculated physicochemical descriptors and pharmacokinetic properties are within the expected range. 9H-thioxanthene drugs 1-4 were subjected to molecular docking to determine their molecular interactions at the active sites of VEGFR-2 and COX-2. The molecular docking study revealed that all four 9H-thioxanthene drugs 1-4 were able to target VEGFR-2 and COX-2. In the future, these findings will be greatly favorable in augmenting the utility of the development of the investigated drugs 1-4 for cancer therapeutics specifically targeting VEGFR-2 and COX-2. © 2022 by the authors. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
... While we use binding of DNA walkers as one illustration of the usefulness of our proposed theory, the scope of our theory extends much further than this example. The spread of infections in animal motion from home ranges [19], the kinetics of V-DJ recombination in chromatin [23], the modulation of protein-ligand activity through tethering of binding agents [24], and molecular detection through tethered particle sandwich assays [25], FIG. 1. Probability density Q(r) of observing two random walkers a distance r apart in d = 1 (dashed lines) and d = 2 (solid lines) for = 0. For d = 2, Q → 0 as r → 0 due to vanishing entropy (Jacobian factor) with r in dimensions higher than one. ...
Diffusion-mediated binding of molecules under the influence of discrete spatially confining potentials is a commonly encountered scenario in systems subjected to explicit fields or implicit fields arising from tethering restraints. Here, we derive analytical expressions for the mean binding time of two random walkers geometrically confined by means of two harmonic potentials in one- and two-dimensional systems, which show excellent agreement with Brownian dynamics simulations. As a demonstration of its utility, we use this theory to maximize the communication speed in existing DNA walkers, obtaining quantitative agreement with previously reported experimental findings. The analytical expressions derived in this paper are broadly applicable to diverse systems, providing ways to characterize communication processes and optimize the rate of signal propagation for sensing and computing applications at the nanoscale.
... Our virtual screening analysis showed that cordycepin and geraniol are promising ligands as they established hydrogen bonds with the reported active site residues of these receptors. Hydrogen bonding of compounds brings disturbance in the structure and function of protein target which ultimately leads to their inhibition as evident from these studies [16,[42][43][44][45] . Moreover, we also determined the toxic side effects and lethal dose of these compounds to ensure safety and dose calibration during in vitro experimentations to procure maximal affectivity against OA. ...
... It is therefore desirable to construct modular biosensor platforms that can be easily repurposed to detect different protein targets of interest. Modular systems have been developed to detect antibodies [7][8][9] and small molecules 10,11 , but general protein sensors are a bigger challenge given the great diversity of protein structures, sizes and oligomerization states, and approaches such as semisynthetic protein platforms [12][13][14] , or calmodulin switches 15,16 , usually require considerable screening to find potential candidates owing to limited predictability 17 . ...
Naturally occurring protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications¹, but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest². The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; binding of the analyte of interest drives switching from the closed to the open state. Because the sensor is based purely on thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the IgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-19³, we used the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder⁴, has a limit of detection of 15 pM and a signal over background of over 50-fold. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.
... In a study, a bioluminescent sensor with a smartphone as a device, named LUMABS (LUMinescent AntiBody Sensor), designed to recognize different antibody molecules in this matrix, was reported (Arts et al., 2016). NanoLuc is a powerful blue fluorescent luciferase that plays an important role in the detection of small doses of therapeutic drugs (Griss et al., 2014;Schena et al., 2015). In order to meet the requirements of bioluminescence resonance energy transfer, the authors selected another monomer green fluorescent protein, mNeonGreen, as the receptor. ...
The emergence of the smartphones has brought extensive changes to our lifestyles, from communicating with one another, to shopping and enjoyment of entertainment, and from studying to functioning at the workplace (and in the field). At the same time, this portable device has also provided new possibilities in scientific research and applications. Based on the growing awareness of good health management, researchers have coupled health monitoring to smartphone sensing technologies. Along the way, there have been developed a variety of smartphone-based optical detection platforms for analyzing biological samples, including standalone smartphone units and integrated smartphone sensing systems. In this review, we outline the applications of smartphone-based optical sensors for biosamples. These applications focus mainly on three aspects: Microscopic imaging sensing, colorimetric sensing and luminescence sensing. We also discuss briefly some limitations of the current state of smartphone-based spectroscopy and present prospects of the future applicability of smartphone sensors.
