Binding Proteomics Analysis

Elucidating Mechanisms of Chemical Toxicity through Target Protein Identification

Toxicology is the science of understanding the adverse effects of diverse chemicals, including pharmaceuticals, cosmetics and consumer products, natural toxins, pesticides, and environmental contaminants, and translating this knowledge into safety assessment.

Organ toxicity and other biological responses caused by chemicals can be detected through animal experiments, cell-based assays, histopathological examination, and omics analyses. However, identifying which proteins a chemical interacts with in vivo and how those interactions lead to toxicity remains a major challenge in toxicology.

Mammals, including humans and experimental animals, possess more than 20,000 proteins. Experimentally screening all of these proteins to identify potential molecular targets of a given chemical is extremely difficult in terms of time, technical feasibility, and cost. This challenge is particularly important for environmental contaminants without known pharmacological targets and for chemicals associated with unexpected adverse health effects. In such cases, identifying potential toxicological targets is an essential first step toward understanding the mechanism of toxicity.

The Need for Toxicological Target Discovery: Puberulic Acid-Associated Kidney Injury as an Example

Recent health incidents involving red yeast rice dietary supplements in Japan drew attention to puberulic acid as a compound suspected of contributing to severe kidney injury. However, little was known about which proteins puberulic acid may interact with or how such interactions could contribute to nephrotoxicity.

This example illustrates a broader challenge in toxicology: even when a chemical becomes a major public health concern, its molecular targets and mechanisms of toxicity may remain poorly understood.

A new analytical framework is therefore needed to rapidly prioritize candidate target proteins and guide subsequent experimental validation.

What Is Binding Proteomics?

Our laboratory has developed a computational framework that uses AlphaFold-predicted protein structures to perform comprehensive molecular docking simulations across the structural proteome of a given species.

In this analysis, chemical compounds are computationally screened against more than 20,000 protein structures registered for individual species, such as humans and mice, in the AlphaFold Protein Structure Database. Proteins are then ranked according to their predicted potential for interaction with the compound of interest.

In addition, functional annotation and enrichment analyses of highly ranked candidate proteins are used to explore biological pathways and mechanistic hypotheses that may be affected by chemical binding.

We refer to this proteome-wide strategy for identifying candidate protein targets of chemicals as Binding Proteomics.

Key Features of Binding Proteomics Analysis

Comprehensive Target Screening

Rather than restricting analysis to a single known target, Binding Proteomics searches across the entire structural proteome to identify potential target proteins.

Cross-Species Applicability

The method can be applied not only to humans and laboratory animals, but also to wildlife species and microorganisms, provided that sufficient protein structural information is available.

Hypothesis Generation for Mechanistic Toxicology

For chemicals whose mechanisms of toxicity are poorly understood, Binding Proteomics can identify candidate target proteins and associated biological pathways for subsequent experimental validation.

Applications in Predictive Toxicology

Potential applications include the identification of off-target proteins for drug candidates, mechanistic evaluation of environmental contaminants, and assessment of interspecies differences in chemical susceptibility.

Importantly, molecular docking scores are theoretical indicators of potential interactions based on structural information. They do not directly demonstrate physiological effects or toxic outcomes.

Binding Proteomics is therefore intended as an exploratory platform for efficiently prioritizing candidate targets that should be investigated through experimental toxicology.

Identification of Candidate Mechanisms for Puberulic Acid-Associated Nephrotoxicity

To evaluate the utility of this approach, our laboratory applied Binding Proteomics analysis to puberulic acid, a compound suspected of contributing to severe kidney injury associated with red yeast rice-related health incidents in Japan.

Comprehensive molecular docking was performed against the AlphaFold-predicted structural proteomes of both humans and mice. The analysis identified sodium/myo-inositol cotransporter 2 (SLC5A11) as a highly ranked candidate target in both species.

SLC5A11 is involved in cellular protection against hyperosmotic stress through myo-inositol uptake in the kidney. Based on this biological function, our findings suggest that disruption of renal osmoregulatory mechanisms may be involved in puberulic acid-associated nephrotoxicity.

This work was published as an original research article in the peer-reviewed international journal The Journal of Toxicological Sciences.

Original Research Article

Comprehensive molecular docking on the AlphaFold-predicted protein structure proteome: identifying target protein candidates for puberulic acid
Teppei Hayama, Rin Sugawara, Ryo Kamata, Masakazu Sekijima, Kazuki Takeda
The Journal of Toxicological Sciences, 50(7), 309–324, 2025.

DOI:
https://doi.org/10.2131/jts.50.309

Publicly Available Analysis Pipeline

The analytical pipeline developed in this study, reAlldock, is publicly available on GitHub.

Using a GPU-equipped computational environment, the pipeline enables users to retrieve protein structures for selected species from the AlphaFold Database and perform structural proteome-wide molecular docking analyses for compounds of interest.

GitHub Repository: reAlldock
https://github.com/toxtoxcat/reAlldock

Collaborative and Contract Research Opportunities

Binding Proteomics analysis can be applied to a broad range of toxicological and pharmacological questions, including target discovery for chemicals with unknown mechanisms of toxicity, off-target screening of drug candidates, mechanistic evaluation of environmental contaminants, and comparative assessment of chemical susceptibility across animal species.

Our laboratory has experience in collaborative research with academic institutions in Japan and overseas, as well as commissioned analyses for industrial companies.

We welcome inquiries regarding collaborative research and contract analyses using the Binding Proteomics platform.

Examples of Potential Research Applications

• Identification of candidate protein targets for novel or known chemicals
• Prediction of potential off-target proteins for pharmaceutical candidates
• Mechanistic hypothesis generation for environmental contaminants and natural toxins
• Comparative analysis of potential target interactions among humans, experimental animals, and wildlife species
• Integrated mechanistic analyses combining molecular docking, molecular dynamics simulations, and enrichment analyses

Contact

Laboratory of Toxicology
School of Veterinary Medicine, Kitasato University
Kazuki Takeda, D.V.M., Ph.D.
Email: takeda{@}vmas.kitasato-u.ac.jp

Research Highlights

Research findings related to Binding Proteomics have been highly recognized through student presentations at scientific meetings.

Rin Sugawara

Rin Sugawara, then a fifth-year undergraduate student, received the Best Presentation Award at the 7th Meeting on Mechanisms of Drug-Induced Toxicity of the Japanese Society of Toxicology in January 2025 for her presentation on the development and evaluation of the Binding Proteomics platform.

Teppei Hayama

Teppei Hayama, a first-year doctoral student, received the American Chemical Society Award at the 4th Joint Conference on Environmental Chemicals in 2025 for his presentation applying Binding Proteomics analysis to puberulic acid.

He was also selected for an award for his presentation on Binding Proteomics analysis at the 2025 Meeting on Environmental Response and Xenobiotic-Metabolizing Enzymes.

International Presentation at the Society of Toxicology Annual Meeting

In March 2026, Kazuki Takeda delivered an invited presentation on the application of Binding Proteomics to mechanistic toxicology and cross-species risk assessment at the 65th Annual Meeting of the Society of Toxicology (SOT), as part of the Global Session:

Structure-Based Toxicology in the AlphaFold Era: Emerging Opportunities and Unmet Challenges

Presentation Title:
Binding-Proteomics in the AlphaFold Era: Scalable Proteome-Wide Docking for Mechanistic Toxicology and Cross-Species Risk Assessment

This presentation highlighted the potential of proteome-wide structural screening to support the identification of toxicological target proteins, mechanistic interpretation of adverse effects, and prediction of chemical susceptibility across species.