Development of a Microscale Thermophoresis-Based Method for Screening and Characterizing Inhibitors of the Methyl-Lysine Reader Protein MRG15

Feoli, Alessandra; Pisapia, Vincenzo; Viviano, Monica; Castellano, Sabrina; Bartoschik, Tanja; Sbardella, Gianluca

doi:10.1177/2472555220949166

Cited by 5 publications

(3 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although it offers several advantages, such as fast, reliable, and label-free measurements, one cannot ignore that the necessity of surface immobilization remains a major challenge. Besides SPR, the significance of MST also emerges. − In contrast to SPR or ITC, thermodynamic properties cannot be analyzed using MST with a single measurement but require several measurements at different temperatures . However, especially in early drug discovery, this sample-saving method can be of great benefit.…”

mentioning

confidence: 99%

An Optimized Microscale Thermophoresis Method for High-Throughput Screening of DNA Methyltransferase 2 Ligands

Zimmermann

Schwickert

Meidner

et al. 2022

ACS Pharmacol. Transl. Sci.

View full text Add to dashboard Cite

Developing methyltransferase inhibitors is challenging, since most of the currently used assays are time-consuming and cost-intensive. Therefore, efficient, fast, and reliable methods for screenings and affinity determinations are of utmost importance. Starting from a literature-known fluorescent S-adenosylhomocysteine derivative, 5-FAM-triazolyl-adenosyl-Dab, developed for a fluorescence polarization assay to investigate the histone methyltransferase mixed-lineage leukemia 1, we herein describe the applicability of this compound as a fluorescent tracer for the investigation of DNA-methyltransferase 2 (DNMT2), a human RNA methyltransferase. Based on these findings, we established a microscale thermophoresis (MST) assay for DNMT2. This displacement assay can circumvent various problems inherent to this method. Furthermore, we optimized a screening method via MST which even indicates if the detected binding is competitive and gives the opportunity to estimate the potency of a ligand, both of which are not possible with a direct binding assay.

show abstract

mentioning

confidence: 99%

An Optimized Microscale Thermophoresis Method for High-Throughput Screening of DNA Methyltransferase 2 Ligands

Zimmermann

Schwickert

Meidner

et al. 2022

ACS Pharmacol. Transl. Sci.

View full text Add to dashboard Cite

show abstract

“…The movement of biomolecules strictly depends on charge, mass, and hydration shell, and when a binding event occurs, at least one of these three parameters is affected, leading to an alteration of thermophoretic movement. These changes in thermophoretic mobility are used to estimate K D values [11u,21] …”

Section: Resultsmentioning

confidence: 99%

DMSO‐Related Effects on Ligand‐Binding Properties of Lysine Methyltransferases G9a and SETD8

Feoli,

Sarno,

Castellano

et al. 2024

ChemBioChem

Self Cite

View full text Add to dashboard Cite

Being the standard solvent for preparing stock solutions of compounds for drug discovery, DMSO is always present in assay buffers in concentrations ranging from 0.1% to 5% (v/v). Even at the lowest concentrations, DMSO‐containing solutions can have significant effects on individual proteins and possible pitfalls cannot be eliminated. Herein, we used two protein systems, the lysine methyltransferases G9a/KMT1C and SETD8/KMT5A, to study the effects of DMSO on protein stability and on the binding of the corresponding inhibitors, using different biophysical methods such as nano Differential Scanning Fluorimetry (nanoDSF), Differential Scanning Fluorimetry (DSF), microscale thermophoresis (MST), and surface plasmon resonance (SPR), all widely used in drug discovery screening campaigns. We demonstrated that the effects of DMSO are protein‐ and technique‐dependent and cannot be predicted or extrapolated on the basis of previous studies using different proteins and/or different assays. Moreover, we showed that the application of orthogonal biophysical methods can lead to different binding affinity data, thus confirming the importance of using at least two different orthogonal assays in screening campaigns. This variability should be taken into account in the selection and characterization of hit compounds, in order to avoid data misinterpretation.

show abstract

“…To overcome the limits of the available screening methods, an appropriate approach should combine the use of different inhibition assays with the evaluation of the direct binding of potential inhibitors with NOX isoforms, applying different assays. , This multiple approach allows one to define genuine inhibition of the proteins caused by functional binding of compounds, avoiding molecules responsible for nonspecific inhibition. Additionally, the application of orthogonal biophysical methods to evaluate interactions is a widely reported strategy, which could help in the validation of obtained binding data, allowing the identification of real and robust inhibitors. An example of a successful application of this combined approach has been recently reported by Mattevi and co-workers, who demonstrated that it can be exploited as a model to continue the research of inhibitors in this field.…”

Section: Conclusion and Future Perspectivementioning

confidence: 99%

NADPH Oxidases: From Molecular Mechanisms to Current Inhibitors

Cipriano,

Viviano,

Feoli

et al. 2023

J. Med. Chem.

Self Cite

View full text Add to dashboard Cite

NADPH oxidases (NOXs) form a family of electrontransporting membrane enzymes whose main function is reactive oxygen species (ROS) generation. Strong evidence suggests that ROS produced by NOX enzymes are major contributors to oxidative damage under pathologic conditions. Therefore, blocking the undesirable actions of these enzymes is a therapeutic strategy for treating various pathological disorders, such as cardiovascular diseases, inflammation, and cancer. To date, identification of selective NOX inhibitors is quite challenging, precluding a pharmacologic demonstration of NOX as therapeutic targets in vivo. The aim of this Perspective is to furnish an updated outlook about the small-molecule NOX inhibitors described over the last two decades. Structures, activities, and in vitro/in vivo specificity are discussed, as well as the main biological assays used. ■ SIGNIFICANCE• ROS produced by NOXs are major contributors to oxidative damage in pathologic conditions. • Therefore, blocking the undesirable actions of these enzymes is a therapeutic strategy for treating various pathological disorders. • The aim of this Perspective is to provide insight on NOX proteins as targets and an overview and update on the current status of NOX inhibitors, including challenges and potential strategic directions for future progress in the field.

show abstract

Development of a Microscale Thermophoresis-Based Method for Screening and Characterizing Inhibitors of the Methyl-Lysine Reader Protein MRG15

Cited by 5 publications

References 40 publications

An Optimized Microscale Thermophoresis Method for High-Throughput Screening of DNA Methyltransferase 2 Ligands

An Optimized Microscale Thermophoresis Method for High-Throughput Screening of DNA Methyltransferase 2 Ligands

DMSO‐Related Effects on Ligand‐Binding Properties of Lysine Methyltransferases G9a and SETD8

NADPH Oxidases: From Molecular Mechanisms to Current Inhibitors

Contact Info

Product

Resources

About