Discovery of New Inhibitors of eEF2K from Traditional Chinese Medicine Based on In Silico Screening and In Vitro Experimental Validation

Abstract

Eukaryotic elongation factor 2 kinase (eEF2K) is a highly conserved α kinase and is increasingly considered as an attractive therapeutic target for cancer as well as other diseases. However, so far, no selective and potent inhibitors of eEF2K have been identified. In this study, pharmacophore screening, homology modeling, and molecular docking methods were adopted to screen novel inhibitor hits of eEF2K from the traditional Chinese medicine database (TCMD), and then cytotoxicity assay and western blotting were performed to verify the validity of the screen. Resultantly, after two steps of screening, a total of 1077 chemicals were obtained as inhibitor hits for eEF2K from all 23,034 compounds in TCMD. Then, to verify the validity, the top 10 purchasable chemicals were further analyzed. Afterward, Oleuropein and Rhoifolin, two reported antitumor chemicals, were found to have low cytotoxicity but potent inhibitory effects on eEF2K activity. Finally, molecular dynamics simulation, pharmacokinetic and toxicological analyses were conducted to evaluate the property and potential of Oleuropein and Rhoifolin to be drugs. Together, by integrating in silico screening and in vitro biochemical studies, Oleuropein and Rhoifolin were revealed as novel eEF2K inhibitors, which will shed new lights for eEF2K-targeting drug development and anticancer therapy.

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“…Western blotting was performed according to previously described methods with some modifications ( Fu et al., 2022 ). Briefly, cells or tissue powders were lysed using RIPA lysis buffer (IN-WB001, Invent, Plymouth, USA) with 1 mmol/L PMSF (P0100, Solarbio Technology Co., Beijing, China).…”

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“…Western blot analysis was performed as previously described [ 18 ]. Briefly, frozen liver and adipose tissues from 3 chickens slaughtered per group were lysed using RIPA lysis buffer (IN-WB001, Invent, Plymouth, MA, USA) with 1 mM PMSF (P0100, Solarbio Technology Co., Beijing, China).…”

eEF2K Inhibitor Design: The Progression of Exemplary Structure-Based Drug Design

“…Though not encompassing, we describe a general workflow for structure-based drug design and the optimization process ( Figure 3 ). A recent example of drug discovery targeting eEF2K, which used a computational screening pipeline with a database of traditional Chinese medicines, identified several antitumor chemicals with low cytotoxicity [ 67 ].…”