Chemoproteomics for Covalent Drug Discovery
Covalent drugs bind to target protein irreversibly to form a stable covalent bond, which leads to enhanced potency, prolonged duration of action, and the ability to target proteins that have been considered "undruggable". However, the strong reactivity poses a significant risk of indiscriminately binding to other proteins, resulting in unforeseen side effects. Chemoproteomics (also known as chemical proteomics) is a powerful solution that can overcome the complexity of covalent drug development. The core of chemical proteomics is utilizing chemical probes and mass spectrometry to identify and quantify the proteins that a compound interacts with. One of the most significant advantages of this approach is the comprehensive identification of the on- and off-targets. This is crucial for the early evaluation of candidate compounds and the optimization of lead molecules.
Chemical proteomics plays a key role in the rational design and optimization of the "warhead", which is the reactive electrophilic group responsible for forming the covalent bond. By systematically profiling the different warheads in a library against the proteome, it is possible to select those warheads that exhibit the desired reactivity and selectivity for the target protein while minimizing interactions with other cellular nucleophiles. Furthermore, the quantitative ability of chemical proteomics provides another critical advantage. By employing techniques such as tandem mass tags (TMT) or label-free quantification method, the extent of candidate interactions with both intended targets and off-targets across varying concentrations can be accurately measured, which enables the assessment of candidate efficacy and selectivity within a physiologically relevant context.
Advantages of Chemoproteomic Technology
| Technologies |
Protein Purification |
Pre-screening Cost |
Target Screening Throughput |
Quantifiability |
Chemical Space |
Non-specific Exclusion |
| Chemoproteomic Technology |
No Required |
Low |
Multiple |
Yes |
Unlimited |
Strong |
| DNA-encoded Molecular Library Screening Technology |
Required |
High |
Single |
No |
Limited (Compatible) |
Weak |
| Affinity Mass Spectrometry |
Required |
High |
Single |
No |
Unlimited |
Weak |
| Surface Plasmon Resonance Screening Technology |
Required |
High |
Single |
Yes |
Unlimited |
Moderate |
Covalent Compound Discovery Platform
Amerigo Scientific provides a covalent drug discovery platform to discover small molecules that covalently bind to the thiol group of cysteine residues in protein targets in living cells. Different from the traditional single-target screening strategy, our platform based on chemical proteomics can quantitatively analyze the interactions between electrophilic fragments and almost all proteins in living cells with the resolution of amino acid residues. Our technology platform can cover over 10,000 proteins and approximately 40,000 cysteine sites from several mammalian cell lines, all of which are potential drug binding sites.
Compound Screening Workflow
Figure 1. The workflow for screening covalent compounds based on DIA-ABPP
Platform Features
- Cysteine-targeted Covalent Library
The cysteine-targeted covalent library contains representative mild electrophilic "warheads", such as acrylamide and chloroacrylamide. The library contains approximately 3,000 compounds, with over 80% of the molecular weights ranging from 300 to 500 Da. For most compounds, the Tanimoto index is approximately 0.3, indicating the high diversity of our library.
Our library of protein targets captured by thiol-specific chemical probe encompasses 39,962 cysteine sites from 12,421 proteins, including kinases, phosphatases, ligases, and transcription factors.
Figure 2. The Tanimoto index among compounds in our covalent compound library
Figure 3. The types of targets included in our protein target library
Application Case
As the substrate receptor of two E3 ligases, CRL4DCAF1 and EDVP, DCAF1 plays a crucial role in protein degradation. Several covalent and non-covalent binding compounds targeting the WDR domain of DCAF1 have been developed for targeted protein degradation (TPD).
Through our chemical proteomics platform, Mol-380 was discovered to be covalently bound to DCAF1_C69, highlighting DCAF1_C69 as a potential drug target for TPD. As shown in Figure 5, the binding site of C69 is located near the validated pocket in the WD40 domain, providing a new binding site for the development of PROTACs.
Our platform is designed to discover new ligands for non-druggable targets in living cells (including transcription factors and membrane proteins), accelerating the screening, development and functional exploration of innovative drugs.
Figure 4. DCAF1_C69 target occupancy study
Figure 5. The structures of DCAF1 and a potential PROTAC molecule
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