Characterization of MEKK1 and MEKK2: Protein Expression, Inhibitor Screening and Crystallization
Rautio, Ilari (2025)
2025
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2025-05-08
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202505085007
https://urn.fi/URN:NBN:fi:tuni-202505085007
Tiivistelmä
The mitogen-activated protein kinase (MAPK) signalling pathways are essential for regulating key cellular processes, with mitogen-activated protein kinase kinase kinases 1 and 2 (MEKK1 and MEKK2) playing pivotal roles in cellular stress responses. In this study, we optimized the recombinant expression, purification, and inhibitor screening of the kinase domains of both MEKK1 and MEKK2, overcoming significant challenges related to protein instability and solubility.
Despite MEKK1's inherent structural instability and tendency to aggregate, we successfully optimized the expression and purification of the MEKK1-MBP construct, enabling functional studies and inhibitor screening. The use of the MBP tag to enhance solubility facilitated the production of soluble MEKK1, providing a valuable platform for identifying potential inhibitors. Through screening, we identified one compound, Hit 1, as a moderate inhibitor of MEKK1, demonstrating promising allosteric inhibition. However, several reported inhibitors, such as crizotinib and staurosporine, showed much weaker activity against MEKK1 than previously indicated in kinase profiling databases. This discrepancy highlights the importance of experimental validation, as the actual inhibitory potential of these compounds was significantly lower under our assay conditions. These findings emphasize the need for more detailed structural studies and further screening efforts to discover potent and selective MEKK1 inhibitors.
In contrast, MEKK2 was expressed more efficiently, particularly in insect cells, where fusion with GST improved solubility and stability. This enabled the successful biochemical analysis and inhibitor screening of MEKK2, revealing several promising inhibitors, including lestaurtinib, AZD-7762, CHZ868, and CDK1/2 Inhibitor III, which demonstrated potent inhibitory activity with IC₅₀ values ranging from 0.03 µM to 0.45 µM. Attempts to structurally characterize MEKK2 through crystallization were unsuccessful, likely due to the protein's conformational flexibility and instability. Despite these challenges, our study provides valuable insights into the bio-chemical properties of both MEKK1 and MEKK2, highlights potential small-molecule inhibitors, and lays the groundwork for future studies aimed at refining expression systems, improving structural characterization techniques, and developing therapeutic strategies targeting these kinases.
Despite MEKK1's inherent structural instability and tendency to aggregate, we successfully optimized the expression and purification of the MEKK1-MBP construct, enabling functional studies and inhibitor screening. The use of the MBP tag to enhance solubility facilitated the production of soluble MEKK1, providing a valuable platform for identifying potential inhibitors. Through screening, we identified one compound, Hit 1, as a moderate inhibitor of MEKK1, demonstrating promising allosteric inhibition. However, several reported inhibitors, such as crizotinib and staurosporine, showed much weaker activity against MEKK1 than previously indicated in kinase profiling databases. This discrepancy highlights the importance of experimental validation, as the actual inhibitory potential of these compounds was significantly lower under our assay conditions. These findings emphasize the need for more detailed structural studies and further screening efforts to discover potent and selective MEKK1 inhibitors.
In contrast, MEKK2 was expressed more efficiently, particularly in insect cells, where fusion with GST improved solubility and stability. This enabled the successful biochemical analysis and inhibitor screening of MEKK2, revealing several promising inhibitors, including lestaurtinib, AZD-7762, CHZ868, and CDK1/2 Inhibitor III, which demonstrated potent inhibitory activity with IC₅₀ values ranging from 0.03 µM to 0.45 µM. Attempts to structurally characterize MEKK2 through crystallization were unsuccessful, likely due to the protein's conformational flexibility and instability. Despite these challenges, our study provides valuable insights into the bio-chemical properties of both MEKK1 and MEKK2, highlights potential small-molecule inhibitors, and lays the groundwork for future studies aimed at refining expression systems, improving structural characterization techniques, and developing therapeutic strategies targeting these kinases.