scholarly journals Sustained enzymatic activity and flow in crowded protein droplets

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Andrea Testa ◽  
Mirco Dindo ◽  
Aleksander A. Rebane ◽  
Babak Nasouri ◽  
Robert W. Style ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Small Molecules ◽  
Collective Behavior ◽  
Living Cells ◽  
Steady States ◽  
Chemical Processes ◽  
Minimal System ◽  
Ph Gradients ◽  
Length Scales ◽  
Far From Equilibrium

AbstractLiving cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium.

scholarly journals Sustained Enzymatic Activity and Flow in Crowded Protein Droplets

2021 ◽  
Author(s):  
Andrea Testa ◽  
Mirco Dindo ◽  
Aleksander A. Rebane ◽  
Babak Nasouri ◽  
Robert W. Style ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Small Molecules ◽  
Collective Behavior ◽  
Living Cells ◽  
Steady States ◽  
Chemical Processes ◽  
Minimal System ◽  
Ph Gradients ◽  
Length Scales ◽  
Far From Equilibrium

Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium.

scholarly journals Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

2020 ◽  
Author(s):  
Maria Kuzikov ◽  
Elisa Costanzi ◽  
Jeanette Reinshagen ◽  
Francesca Esposito ◽  
Laura Vangeel ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Small Molecules ◽  
Drug Target ◽  
Treatment Options ◽  
Cleavage Sites ◽  
X Ray ◽  
Binding Characteristics ◽  
Main Protease ◽  
Ic50 Values

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 uM and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Angs., showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

scholarly journals Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Shaoying Lu ◽  
Jihye Seong ◽  
Yi Wang ◽  
Shiou-chi Chang ◽  
John Paul Eichorst ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Focal Adhesions ◽  
Living Cells ◽  
Dynamic Coordination ◽  
Structural Modulation

scholarly journals Identification of Small Molecule Inhibitors of the Deubiquitinating Activity of the SARS-CoV-2 Papain-Like Protease: in silico Molecular Docking Studies and in vitro Enzymatic Activity Assay

2020 ◽  
Vol 8 ◽  
Author(s):  
Eleni Pitsillou ◽  
Julia Liang ◽  
Katherine Ververis ◽  
Kah Wai Lim ◽  
Andrew Hung ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Enzymatic Activity ◽  
Small Molecules ◽  
Binding Site ◽  
Protein Docking ◽  
Activity Assay ◽  
Epicatechin Gallate ◽  
Inhibitor Binding ◽  
Enzymatic Activity Assay

COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 virus with important political, socio-economic, and public health consequences. Inhibiting replication represents an important antiviral approach, and in this context two viral proteases, the SARS-CoV-2 main and papain-like proteases (PLpro), which cleave pp1a and pp1ab polypeptides, are critical. Along with protease activity, the PLpro possesses deubiquitinating activity, which is important in immune regulation. Naphthalene-based inhibitors, such as the well-investigated GRL-0617 compound, have been shown to possess dual effects, inhibiting both protease and deubiquitinating activity of the PLpro. Rather than binding to the canonical catalytic triad, these type of non-covalent inhibitors target an adjacent pocket, the naphthalene-inhibitor binding site. Using a high-throughput screen, we have previously identified the dietary hypericin, rutin, and cyanidin-3-O-glucoside compounds as potential protease inhibitors targeting the naphthalene-inhibitor binding site. Here, our aim was to investigate the binding characteristics of these compounds to the PLpro, and to evaluate deubiquitinating activity, by analyzing seven different PLpro crystal structures. Molecular docking highlighted the relatively high affinity of GRL-0617 and dietary compounds. In contrast binding of the small molecules was abolished in the presence of ubiquitin in the palm subdomain of the PLpro. Further, docking the small molecules in the naphthalene-inhibitor binding site, followed by protein-protein docking revealed displacement of ubiquitin in a conformation inconsistent with functional activity. Finally, the deubiquitinating activity was validated in vitro using an enzymatic activity assay. The findings indicated that the dietary compounds inhibited deubiquitinase activity in the micromolar range with an order of activity of GRL-0167, hypericin >> rutin, cyanidin-3-O-glucoside > epigallocatechin gallate, epicatechin gallate, and cefotaxime. Our findings are in accordance with mechanisms and potential antiviral effects of the naphthalene-based, GRL-0617 inhibitor, which is currently progressing in preclinical trials. Further, our findings indicate that in particular hypericin, rutin, and cyanidin-3-O-glucoside, represent suitable candidates for subsequent evaluation as PLpro inhibitors.

scholarly journals Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells

2010 ◽  
Vol 107 (5) ◽  
pp. 1870-1875 ◽  
Author(s):  
Alex K. Shalek ◽  
Jacob T. Robinson ◽  
Ethan S. Karp ◽  
Jin Seok Lee ◽  
Dae-Ro Ahn ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Silicon Nanowires ◽  
Mammalian Cells ◽  
Living Cells ◽  
Diverse Range ◽  
Viral Packaging ◽  
Neuronal Progenitor ◽  
Efficient Delivery ◽  
Surface Modified

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell’s membrane and subsequently release surface-bound molecules directly into the cell’s cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology’s potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.

scholarly journals The RASSCF, RASSI, and CASPT2 Methods Used on Small Molecules of Astrophysical Interest

1994 ◽  
Vol 146 ◽  
pp. 338-352
Author(s):  
Per-Åke Malmqvist
Keyword(s):  
Perturbation Theory ◽  
Small Molecules ◽  
Stellar Atmosphere ◽  
Second Order ◽  
Cloud Chemistry ◽  
Complete Active Space ◽  
Active Space ◽  
Computational Quantum Chemistry ◽  
Self Consistent Field ◽  
Far From Equilibrium

To a quantum chemist with no particular background in astrophysics or astronomy, a brief glance at journals and textbooks in these fields shows at least three areas where computational quantum chemistry has had a valuable impact: Interstellar cloud chemistry; stellar atmosphere modelling; and chemistry in extreme conditions, such as at the surface of a neutron star. The first two uses are particularly suitable, since standard methods are directly applicable.For such problems, good calculations of potential energy as well as expectation values and matrix elements of dipole and other operators appears to be in demand. Many electronic states may be involved, at least a broad range of problems involve fairly small molecules, often radicals, and conformation regions far from equilibrium. Such problems are addressed by three methods originated in our laboratory, and known by the acronyms RASSCF (Restricted Active Space Self-Consistent Field, Malmqvist et al. 1990), RASSI (RAS State Interaction) and CASPT2 (Complete Active Space Perturbation Theory to Second Order-Complete Active Space Perturbation Theory to Second Order, Andersson et al. 1990; Andersson et al. 1992).

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