【Liman "Protein Degradation Drug Product Series"】Officially Launched!

Liman Bio's team has been deeply engaged in the development of protein degradation drugs, providing services ranging from target discovery and validation, lead compound discovery and optimization, mechanism of action (MOA) research, and PK/PD. Liman provides high-throughput gene editing products (KO/KI cell lines) and supporting mass spectrometry detection reagents and personalized services to optimize and accelerate the research process. We will provide a complete series of solutions for pharmaceutical companies and basic scientific research clients through a series of products in this field.

Figure 1 Liman's product and service layout in the TPD field

1. Protein ubiquitination and ubiquitinome determine the mechanism of action (MOA) of protein degradation drugs

Ubiquitination of proteins is a modification process mediated by E3 ubiquitin ligase, which degrades target proteins through the ubiquitin-proteasome system (UPS), thereby maintaining cellular homeostasis. In recent years, this important biological process has been utilized to promote the interaction between E3 ubiquitin ligases and proteins of interest (POIs) associated with certain diseases through small molecule compounds (such as PROTACs or molecular glues), inducing ubiquitination and specific degradation of these disease-driving proteins to achieve therapeutic effects. With more and more protein degradation drug molecules entering preclinical research and clinical trials, ubiquitination proteomics, through the specific identification and quantification of ubiquitinated POIs, provides important information on the degree and dynamic changes of ubiquitination. This helps us understand the mechanism of action and efficacy of these compounds and provides direct data support for optimizing structure-activity relationships (SAR). In addition, ubiquitination proteomics can also identify potential off-target effects of targeted protein degraders by analyzing the ubiquitination patterns of cell or tissue samples, thereby reducing the risk of adverse clinical reactions caused by off-target effects.

Figure 2 PROTAC degrader working principle

2. Liman's self-developed K-diGly antibody

Ubiquitination refers to the process in which the ε-amino group of lysine (K) in the substrate protein forms an amide bond with the carboxyl group of the glycine at the diGly end of the ubiquitin molecule. After trypsin cleaves arginine (R) and lysine (K), the characteristic sequence K-GG is produced. Because ubiquitination modifications usually have low abundance and a wide dynamic range, enrichment of modified peptides is required before detection for mass spectrometry identification. To this end, we first performed animal immunization of ubiquitinated peptides and successfully obtained the ELEMAb™ K-ε-GG Antibody against ubiquitination modification. Subsequently, the enrichment efficiency of standardized ubiquitinated modified peptides was evaluated using SpikeMix™ PTM-Kit 47-Lys (GG).

3. High-repeatability ubiquitination proteomics

More than 96.54% of ubiquitinated modified proteins and 93.51% of ubiquitinated modified peptides could be repeatedly identified, indicating that the ELEMAb™ K-ε-GG Antibody has very good repeatability in ubiquitination modification identification. This result shows the reliability and stability of this antibody in identifying and enriching ubiquitination modifications, providing a solid foundation for subsequent research.

Figure 5 ELEMAb™K-ε-GG Antibody identification repeatability evaluation

4. Ubiquitination proteomics confirms the mechanism of action of degraders

Lenalidomide is a drug with significant clinical efficacy against multiple myeloma and other B-cell tumors. To further analyze the mechanism of action of lenalidomide, researchers used ubiquitination proteomics to find that lenalidomide leads to increased ubiquitination levels of IKZF1 and IKZF3. In addition, through whole proteome analysis, it was found that lenalidomide can reduce the abundance of IKZF1 and IKZF3. IKZF1 and IKZF3 are key transcription factors in multiple myeloma. This discovery reveals the mechanism by which lenalidomide exerts its anti-tumor effect by regulating the ubiquitination and degradation of these transcription factors.

Figure 6 Ubiquitination proteomics reveals the mechanism of action of lenalidomide

References

1. Targeted protein degradation mechanisms. Drug Discov Today Technol. 2019 Apr;31:53-60.

2. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 2014 Jan 17;343(6168):301-5.