Functional Label-Free Assays for Characterizing the in Vitro Mechanism of Action of Small Molecule Modulators of Capsid Assembly

Biochemistry ◽  
2015 ◽  
Vol 54 (13) ◽  
pp. 2240-2248 ◽  
Author(s):  
Latesh Lad ◽  
Sheila Clancy ◽  
David Koditek ◽  
Melanie H. Wong ◽  
Debi Jin ◽  
...  
Keyword(s):  
Small Molecule ◽  
Mechanism Of Action ◽  
Capsid Assembly ◽  
Label Free ◽  
Small Molecule Modulators

scholarly journals Therapeutic targeting of measles virus polymerase with ERDRP-0519 suppresses all RNA synthesis activity

2021 ◽  
Vol 17 (2) ◽  
pp. e1009371
Author(s):  
Robert M. Cox ◽  
Julien Sourimant ◽  
Mugunthan Govindarajan ◽  
Michael G. Natchus ◽  
Richard K. Plemper
Keyword(s):  
Small Molecule ◽  
Mechanism Of Action ◽  
Measles Virus ◽  
Rna Synthesis ◽  
De Novo ◽  
Ligand Docking ◽  
Label Free ◽  
Binding Characteristics ◽  
Polymerase Complex

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA.

scholarly journals Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

2020 ◽  
Vol 48 (20) ◽  
pp. e120-e120 ◽  
Author(s):  
Obtin Alkhamis ◽  
Weijuan Yang ◽  
Rifat Farhana ◽  
Haixiang Yu ◽  
Yi Xiao
Keyword(s):  
Ligand Binding ◽  
Small Molecule ◽  
High Throughput ◽  
Dna Aptamer ◽  
Label Free ◽  
General Applicability ◽  
Gold Standard Method ◽  
Characterization Methods ◽  
Increased Risk

Abstract In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion.

scholarly journals Mechanism-based sirtuin enzyme activation

2015 ◽  
Author(s):  
Xiangying Guan ◽  
Alok Upadhyay ◽  
Sudipto Munshi ◽  
Raj Chakrabarti
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
False Positives ◽  
Enzyme Activation ◽  
Label Free ◽  
Multiple Substrates ◽  
Dependent Protein ◽  
Biophysical Techniques ◽  
Catalyzed Reactions ◽  
Small Molecule Modulators

AbstractSirtuin enzymes are NAD+-dependent protein deacylases that play a central role in the regulation of healthspan and lifespan in organisms ranging from yeast to mammals. There is intense interest in the activation of the seven mammalian sirtuins (SIRT1-7) in order to extend mammalian healthspan and lifespan. However, there is currently no understanding of how to design sirtuin-activating compounds beyond allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates. Moreover, across all families of enzymes, only a dozen or so distinct classes of non-natural small molecule activators have been characterized, with only four known modes of activation among them. None of these modes of activation are based on the unique catalytic reaction mechanisms of the target enzymes. Here, we report a general mode of sirtuin activation that is distinct from the known modes of enzyme activation. Based on the conserved mechanism of sirtuin-catalyzed deacylation reactions, we establish biophysical properties of small molecule modulators that can in principle result in enzyme activation for diverse sirtuins and substrates. Building upon this framework, we propose strategies for the identification, characterization and evolution of hits for mechanism-based enzyme activating compounds. We characterize several small molecules reported in the literature to activate sirtuins besides SIRT1, using a variety of biochemical and biophysical techniques including label-free and labeled kinetic and thermodynamic assays with multiple substrates and protocols for the identification of false positives. We provide evidence indicating that several of these small molecules reported in the published literature are false positives, and identify others as hit compounds for the design of compounds that can activate sirtuins through the proposed mechanism-based mode of action.

scholarly journals Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

2007 ◽  
Vol 282 (49) ◽  
pp. 35666-35678 ◽  
Author(s):  
Adam Shutes ◽  
Cercina Onesto ◽  
Virginie Picard ◽  
Bertrand Leblond ◽  
Fabien Schweighoffer ◽  
...  
Keyword(s):  
Small Molecule ◽  
Mechanism Of Action ◽  
Rho Gtpase ◽  
Human Cancer ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.

scholarly journals Biological and catalytic functions of sirtuin 6 as targets for small-molecule modulators

2020 ◽  
Vol 295 (32) ◽  
pp. 11021-11041 ◽  
Author(s):  
Mark A. Klein ◽  
John M. Denu
Keyword(s):  
Small Molecule ◽  
Genome Stability ◽  
Tumor Development ◽  
Molecular Architecture ◽  
Cellular Functions ◽  
Physiological Level ◽  
Biological Insight ◽  
Small Molecule Modulators ◽  
Catalytic Functions

Sirtuin 6 (SIRT6) is a nuclear NAD+-dependent deacetylase of histone H3 that regulates genome stability and gene expression. However, nonhistone substrates and additional catalytic activities of SIRT6, including long-chain deacylation and mono-ADP-ribosylation of other proteins, have also been reported, but many of these noncanonical roles remain enigmatic. Genetic studies have revealed critical homeostatic cellular functions of SIRT6, underscoring the need to better understand which catalytic functions and molecular pathways are driving SIRT6-associated phenotypes. At the physiological level, SIRT6 activity promotes increased longevity by regulating metabolism and DNA repair. Recent work has identified natural products and synthetic small molecules capable of activating the inefficient in vitro deacetylase activity of SIRT6. Here, we discuss the cellular functions of SIRT6 with a focus on attributing its catalytic activity to its proposed biological functions. We cover the molecular architecture and catalytic mechanisms that distinguish SIRT6 from other NAD+-dependent deacylases. We propose that combining specific SIRT6 amino acid substitutions identified in enzymology studies and activity-selective compounds could help delineate SIRT6 functions in specific biological contexts and resolve the apparently conflicting roles of SIRT6 in processes such as tumor development. We further highlight the recent development of small-molecule modulators that provide additional biological insight into SIRT6 functions and offer therapeutic approaches to manage metabolic and age-associated diseases.

scholarly journals Label-Free Detection of G Protein–SNARE Interactions and Screening for Small Molecule Modulators

2011 ◽  
Vol 3 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Christopher A. Wells ◽  
Katherine M. Betke ◽  
Craig W. Lindsley ◽  
Heidi E. Hamm
Keyword(s):  
G Protein ◽  
Small Molecule ◽  
Label Free ◽  
Label Free Detection ◽  
Small Molecule Modulators

Using NanoBit complementation and TR FRET to Study the Mechanism of Action of Small‐molecule Modulators of CXCR2

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Desislava Nikolaeva Nesheva ◽  
Steven Charlton ◽  
Sheilesh Mistry ◽  
Nicholas Holliday
Keyword(s):  
Small Molecule ◽  
Mechanism Of Action ◽  
Small Molecule Modulators

scholarly journals Identification of Small-Molecule Inhibitors of the Ribonuclease H2 Enzyme

2013 ◽  
Vol 18 (5) ◽  
pp. 610-620 ◽  
Author(s):  
Rachel White ◽  
Barbara Saxty ◽  
Jonathan Large ◽  
Catherine A. Kettleborough ◽  
Andrew P. Jackson
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Genetic Disorder ◽  
Rnase H ◽  
Cellular Functions ◽  
Small Molecule Modulators ◽  
Hit Validation ◽  
Inhibitor Compounds ◽  
Rnase H2

Ribonuclease H2 (RNase H2) is a nuclease that specifically hydrolyzes RNA residues in RNA-DNA hybrids. Mutations in the RNase H2 enzyme complex have been identified in the genetic disorder Aicardi-Goutières syndrome (AGS), which has similarities to the autoimmune disease systemic lupus eryrthrematosis (SLE). The RNase H2 enzyme has also been recently implicated as a key genome surveillance enzyme. Therefore, small-molecule modulators of RNase H2 activity may have utility in therapeutics and as tools to investigate the cellular functions of RNase H2. A fluorescent quench assay, measuring cleavage of an RNA-DNA duplex substrate by recombinant RNase H2, was developed into a high-throughput format and used to screen a 48 560 compound library. A hit validation strategy was subsequently employed, leading to the identification of two novel inhibitor compounds with in vitro nanomolar range inhibition of RNase H2 activity and >100-fold selectivity compared with RNase H type 1. These compounds are the first small-molecule inhibitors of RNase H2 to be reported. They and their derivatives should provide the basis for the development of tool compounds investigating the cellular functions of the RNase H2 enzyme, and, potentially, for pharmacological manipulation of nucleic acid–mediated immune responses.

scholarly journals Progress in the Discovery of Small-Molecule Inhibitors of Bromodomain–Histone Interactions

2011 ◽  
Vol 16 (10) ◽  
pp. 1170-1185 ◽  
Author(s):  
Chun-wa Chung ◽  
Jason Witherington
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Ligand Selectivity ◽  
Small Molecule Ligands ◽  
Associated Proteins ◽  
In Vitro Systems

Bromodomains are structurally conserved protein modules present in a large number of chromatin-associated proteins and in many nuclear histone acetyltransferases. The bromodomain functions as an acetyl-lysine binding domain and has been shown to be pivotal in regulating protein–protein interactions in chromatin-mediated cellular gene transcription, cell proliferation, and viral transcriptional activation. Structural analyses of these modules in complex with acetyl-lysine peptide ligands provide insights into the molecular basis for recognition and ligand selectivity within this epigenetic reader family. However, there are significant challenges in configuring assays to identify inhibitors of these proteins. This review focuses on the progress made in developing methods to identify peptidic and small-molecule ligands using biophysical label-free and biochemical approaches. The advantage of each technique and the results reported are summarized, highlighting the potential applicably to other reader domains and the caveats in translation from simple in vitro systems to a biological context.

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