Fragment-based Drug Discovery Interaction Analysis

Fragment-based drug discovery represents a departure from traditional high-throughput screening approaches. Instead of screening large libraries of compounds, FBDD involves screening small, low-molecular-weight fragments that possess favorable drug-like properties. These fragments typically exhibit weak binding affinity for the target protein, but their small size provides an advantage in terms of chemical space coverage and efficient screening.

The primary objective of FBDD is to identify fragment hits that can bind to the target protein with high ligand efficiency, i.e., a strong binding affinity relative to their molecular weight. Once identified, these fragments serve as a foundation for the development of larger, more potent compounds through fragment elaboration or merging strategies. Consequently, accurate analysis of fragment-protein interactions is critical for guiding the optimization process and ultimately leading to the discovery of novel therapeutics.

A flowchart of FBDD (Li et al., 2020).

Techniques for Fragment-Protein Interaction Analysis in Creative Proteomics

1. MicroScale Thermophoresis (MST)

MicroScale Thermophoresis (MST) is a powerful biophysical method used for fragment screening and binding affinity determination. In this technique, a fluorescently labeled target protein is titrated with varying concentrations of fragments. Changes in thermophoretic movement of the labeled protein in response to binding events are measured, allowing for the determination of fragment-protein binding affinities. MST provides high sensitivity, excellent reproducibility, and the ability to work with low sample volumes.

By employing MST, Creative Proteomics enables fragment screening at high concentrations, overcoming the limitations imposed by weak fragment affinities. This approach enhances the probability of identifying hits and enables the detection of weak or transient interactions that might be missed by other screening methods. Furthermore, MST is compatible with high solvent contents, making it suitable for screening in DMSO-rich environments.

2. Nano-Differential Scanning Fluorimetry (nanoDSF)

Nano-Differential Scanning Fluorimetry (nanoDSF) is another biophysical method employed by Creative Proteomics for fragment-based drug discovery. This technique utilizes changes in the fluorescence emission of a thermally sensitive dye to monitor protein stability and ligand binding events. By subjecting the protein-fragment mixtures to a thermal ramp, nanoDSF provides information on the thermal stability and conformational changes induced by fragment binding. The method is highly sensitive to weak interactions and can detect binding events even in the presence of high solvent contents.

Creative Proteomics's utilization of nanoDSF allows for high-throughput fragment screening with high fragment concentrations and in solvent-rich conditions. This approach enhances the screening efficiency and accuracy by providing insights into fragment binding and its impact on protein stability.

3. Isothermal Titration Calorimetry (ITC)

Isothermal Titration Calorimetry (ITC) is a label-free biophysical technique that directly measures heat changes to study molecular interactions. In fragment screening and interaction analysis, ITC is widely used to determine the thermodynamic parameters of binding between fragments and proteins, such as the binding constant (Kd), enthalpy change (ΔH), and entropy change (ΔS). By titrating fragments into a solution containing the target protein, ITC measures the heat released or absorbed, providing insights into the affinity and thermodynamic properties of the fragment-protein interaction.

ITC offers the advantage of direct measurement without the need for labeling or fluorescence signals. It can be applied to study various types of interactions, including fragment-protein, ligand-receptor, and protein-protein interactions. Additionally, ITC can reveal the binding mode and binding sites of fragments with proteins.

4. NMR Spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for studying fragment-protein interactions in solution. NMR can provide valuable information regarding the binding site, conformational changes, and dynamics of the protein upon fragment binding. Two commonly employed NMR methods for fragment-based drug discovery are:

• SAR by NMR: Structure-Activity Relationship by NMR involves synthesizing a series of fragments with subtle structural variations and studying their binding interactions with the target protein using NMR. This approach helps identify key binding interactions and guides the optimization process.
• STD-NMR: Saturation Transfer Difference NMR is a technique used to identify fragments that bind to a protein target. STD-NMR measures the exchange of magnetization between protein-bound fragments and the unbound fragments in solution, providing valuable information on fragment binding.

5. X-ray Crystallography

X-ray crystallography is a widely used technique for determining the three-dimensional structure of proteins and their complexes with fragments. This technique provides atomic-level resolution of fragment-protein interactions, allowing researchers to visualize the binding mode and identify critical binding interactions.

By co-crystallizing the protein of interest with fragment hits, X-ray crystallography enables the determination of the complex structure. The resulting structural information is essential for guiding the optimization process, as it provides insights into the binding site, intermolecular contacts, and potential areas for modification.

6. Surface Plasmon Resonance (SPR)

Surface Plasmon Resonance (SPR) is an optical technique used to measure the real-time binding kinetics and affinity between a fragment and its protein target. SPR detects changes in the refractive index at the surface of a sensor chip as a result of fragment-protein interactions.

SPR offers several advantages, including label-free detection, low sample consumption, and the ability to determine kinetic parameters such as association and dissociation rates. It provides valuable quantitative data on the binding strength and kinetics of fragment-protein interactions, aiding in the selection and optimization of fragments for drug development.

7. Computational Modeling and Docking Studies

Computational modeling and docking studies play a pivotal role in fragment-based drug discovery interaction analysis. These techniques involve the use of molecular docking algorithms to predict the binding mode and affinity of fragments with the target protein.

By employing structural information from X-ray crystallography or NMR spectroscopy, computational docking can generate models of fragment-protein complexes and estimate their binding affinities. This information guides the selection and optimization of fragments during the drug discovery process.

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