p38-alpha Inhibition by Rooperol: Docking and Kinase Assay Studies




Estevez Posadas, Blanca

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p38α is a protein kinase with a high importance in cell metabolism due to its role in inflammation, apoptosis, differentiation, and cell cycle regulation. It is activated by a cascade of reactions that starts with cytokines, growth factors, and a variety of cellular stresses. p38α also has a main role in cancer cells, therefore, its inhibition is of great importance for cancer treatments. There have been many attempts to develop inhibitors for p38α, most of them target the conserved ATP active site. Since the inhibitors that target this site compete with the ATP, the efficacy of inhibiting p38α is reduced. Therefore, it is crucial to find molecules that inhibit p38α by targeting allosteric active sites. Rooperol is a natural product obtained from the African potato that has shown inhibitory activity against p38α by targeting the allosteric DRS active site. Nevertheless, this molecule has a short half-life in the human metabolism. Therefore, it is essential to obtain p38α inhibitors from rooperol analogues that last longer in the human metabolism. To measure the inhibition performed by such analogues, it is necessary to standardize an assay to measure the percent inhibition. Nowadays, there are some assays already developed to measure protein kinase enzyme inhibition. One of the most used techniques requires radioactive labeling of ATP with 33P or 32P. This technique provides direct measurement of the incorporation of phosphorous by protein kinase substrates and few sources of interference, but the risk of using radioactive materials leads hazards that must be managed during the reaction and special waste disposal. Therefore, non-radioactive assays are also well used such as fluorescent and luminescent endpoint assays. Nevertheless, these techniques also have some disadvantages such as high interference, and specific requirements of antibody reagents. Hence, there is great necessity to standardize such assays with the specific requirements for each enzyme. In this work, the region(s) of the p38a DRS that are most likely bound by rooperol are investigated. Also, the universal ATP-Glow kinase assay for use on a pipetting robot to determine protein kinase inhibition was miniaturized and adapted. The results obtained in this work are described in two parts. Firstly, the computational 3D modeling. Secondly, the miniaturization and standardization of the ADP-Glow reaction. The 3D modeling was performed using computational software such as AutoDockTools, Vina, and Chimera. This analysis was performed to measure the affinity of rooperol to an active site in p38α close to the DRS site (the alternative groove) in 9 different crystal structures of the p38α enzyme. The predicted affinity results obtained ranged from -5.2 to -6.1 kcal/mol. These results show a moderate affinity between the alternative groove and rooperol. Nevertheless, they are significantly inferior to the results obtained from the DRS site that ranged from -6.5 to -7.3 kcal/mol (results obtained from another member of the research group). Thus, the alternative groove did not show a greater affinity as the DRS site and should not be further explored. In the second part of the results, the changes made to the ADP-Glow reaction to standardize, minimize, and adapt it for HTS were measured by Zfactor, S/N (signal to noise), S/B (signal to base line), and R². The results obtained were 0.9, 430, 141, and 0.981, respectively. These results showed that the standardized assay is excellent.



p38-alpha, rooperol, protein kinases, kinase assay


Estevez Posadas, B. (2021). p38-alpha inhibition by rooperol: Docking and kinase assay studies (Unpublished thesis). Texas State University, San Marcos, Texas.


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