Research articleQM/MM–PB/SA scoring of the interaction strength between Akt kinase and apigenin analogues
Graphical abstract
The rigorous QM/MM–PB/SA is employed to screen for strong Akt binders from a set of apigenin analogues and, consequently, four compounds, namely apigenin, quercetin, gallocatechin and myricetin, are suggested to have high binding potency to Akt active site, which are further solidified by a kinase assay.
Introduction
Flavonoids consist of a large group of polyphenolic compounds and are ubiquitously present in plants. They are synthesized by phenylpropanoid pathway and can be categorized, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones. Over 4000 flavonoids have been identified, many of which occur in fruits, vegetables and beverages (Heim et al., 2002). Flavonoids have been shown to have a wide range of biological and pharmacological activities, including anti-allergic, anti-inflammatory, antioxidant, anti-microbial, anti-cancer and anti-diarrheal activities (Si et al., 2009, Chakravarthi et al., 2009, Taupin, 2009, Zhao et al., 2010, Ruela-de-Sousa et al., 2010, Ohno et al., 2013); the activities and functions depend on their structural class, degree of hydroxylation, other substitutions and conjugations, and degree of polymerization (Kumar and Pandey, 2013).
On of the most biologically important flavonoids is the apigenin, which has been shown to be a very potent anti-cancer compound. It beneficially protects against a wide variety of cancers with high selectivity for cancer cells as opposed to non-cancerous cells (Gupta et al., 2001, Patel et al., 2007, Shukla and Gupta, 2010). It also has a very high safety threshold, and active doses can be gained through consuming a vegetable and fruit rich diet. In vitro studies implicated that the apigenin inhibits the development, proliferation and invasion of tumor cells is to inactivate Akt signaling pathway by binding specifically to the Akt kinase, which triggers apoptosis in human cancer (Kaur et al., 2008). The Akt also known as protein kinase B (PKB), is a human homologue of the viral oncogene v-Akt that plays a key role in multiple cellular processes such as glucose metabolism, cell proliferation, transcription and migration (Fayard et al., 2005). However, the molecular mechanism of Akt–apigenin recognition and interaction still remain largely unexplored to date. The apigenin is a flavonoid derivative that is substituted by three hydroxyl functional groups at positions 4′, 5 and 7 of the basic flavonoid skeleton, namely, flavone. Accumulated data revealed that many flavonoids exert chemopreventive effects through acting at protein kinase signaling pathways, more than as conventional hydrogen-donating antioxidants. Recent studies show that flavonoid inhibitors can bind directly to certain protein kinases such as Fyn JAK, MEK1 and PI3K, and then alter their phosphorylation state to regulate multiple cell signaling pathways in carcinogenesis processes (Hou and Kumamoto, 2010). Calorimetric studies and theoretical investigations have revealed an essential role of substituents in flavonoid’s selectivity and affinity toward their cognate targets; minor change in substituent type, position and/or quantity can address a marked effect on the binding behavior of flavonoid ligands to protein receptors (Saija et al., 1995, Jain et al., 1999, Shi et al., 2011).
The flavonoid skeleton possesses a number of positions at which the substitutions can result in various flavonoid derivatives. Thus, empirical rule has been widely used to perform theoretical analysis of protein–flavonoid interaction and rational design of novel flavonoid ligands that can bind specifically and stably to the protein targets of biological interest. Previously, however, we demonstrated a typical non-additive contribution of hydroxyl substituents to Akt–apigenin affinity by integrating several sophisticated molecule modeling techniques, and we also found that the non-additivity can only be described accurately with rigorous QM/MM–PB/SA approach (Lu et al., 2014). Very recently, Wichapong et al. (2014) compared QM/MM–GB/SA, MM-GB/SA and MM-PB/SA in discriminating between active and inactive kinase inhibitors, and results showed that QM/MM–GB/SA performed much better than normal docking scores or MM-GB/SA and MM-PB/SA in classifying active and inactive inhibitors. In the current study, we attempted to implement QM/MM–PB/SA scoring of the relative binding strength of apigenin and its flavonoid analogues to Akt kinase. Before the analysis we have collected a number of small-molecule Akt inhibitors with known biological activities to test the QM/MM–PB/SA scoring performance, and the obtained results were compared systematically with that derived from empirical scoring functions and MM-PB/SA analysis with or without assistance of molecular dynamics (MD) simulations. The method was later used to computationally estimate the relative binding strength of apigenin and its flavonoid analogues to Akt kinase. We also performed kinase assay to test the Akt inhibitory activity of several highly promising candidates screened with the QM/MM–PB/SA scoring strategy.
Section snippets
Small-molecule Akt inhibitors as well as apigenin and its flavonoid analogues
Akt inhibitor set (Table 1). We collected eight existing small-molecule inhibitors that have been reported previously to possess both high affinity and selectivity toward Akt kinase. The biological activities, i.e., IC50 values, of these inhibitors were determined by standard kinase assay experiment; all of the values are at nanomolar level, showing a strong binding potency to Akt. These compounds are diverse in terms of various structure features they possess, including quinoline and indole
Estimation of Akt–inhibitor affinity at different theoretical levels
Four out of the eight Akt inhibitors listed in Table 1 have already been solved complex crystal structures with Akt kinase. The complex structure models of other inhibitors with Akt were manually modified from these crystal templates using a protocol described above. These crystal structures or coarse-grained structure models were relaxed via atomistic molecular dynamics (MD) simulations with AMBER03 force field (Duan et al., 2003). Because the unbound Akt structure is in the ‘DFG-in’ state
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Nos. 31271272 and 31200602), the Jiangsu University for Advanced Professionals (No. 1281330021) and the China Postdoctoral Science Foundation (No. 2012M521001).
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