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Ligand Binding Sites

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...

The Equilibrium Binding Constant and Binding Strength

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:

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Updated: Jun 21, 2026

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

Published on: February 28, 2025

Scaling covalent ligand discovery through dynamic combinatorial library-versus-proteome screening.

Yuchen Huang^1,2, Lexuan Hou^1,3, Ruiping He⁴

¹Institute of Chemical Biology, Shenzhen Bay Laboratory, Shenzhen, China.

Nature Communications

|June 19, 2026

Summary

This summary is machine-generated.

We developed a new "library-versus-proteome" platform for faster drug discovery. This method screens many compounds simultaneously, identifying potent covalent inhibitors for various enzymes and revealing new biological roles.

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Area of Science:

Chemical Biology
Proteomics
Drug Discovery

Background:

Mass spectrometry-based chemical proteomics offers unbiased assessment of ligand interactions.
Current screening methods are limited by low throughput and reliance on pre-synthesized compound libraries.

Purpose of the Study:

To develop a high-throughput, mechanism-driven platform for real-time ligand selection and optimization.
To establish a "screen first, synthesize later" paradigm for efficient ligand discovery.

Main Methods:

Coupling dynamic combinatorial libraries with activity-based protein profiling in a "library-versus-proteome" approach.
Application to discover covalent inhibitors of serine hydrolases and cysteine-targeting ligands.
Utilizing a gel-based assay for rapid screening of compound analogues.

Main Results:

Increased screening throughput by 10- to 20-fold.
Discovered covalent inhibitors for serine hydrolases (PPME1, ABHD11, PNPLA6) and identified novel roles for PNPLA6.
Achieved proteome-wide EC50 profiling of over 2600 cysteines, yielding inhibitors for multiple targets including VCP.
Uncovered a GPCR-mediated signaling axis influencing the unfolded protein response.
Identified a potent VCP ligand with nanomolar activity and in vivo antitumor efficacy.

Conclusions:

The "library-versus-proteome" screening strategy is a scalable and efficient method for ligand discovery.
This platform accelerates the identification of potent ligands and provides insights into biological pathways.
The approach is versatile, applicable to various target classes including serine hydrolases and cysteine-containing proteins.