Abstract
Pentacyclic Triterpenoids (PTs) and their analogues as well as derivatives are emerging as important drug leads for various diseases. They act through a variety of mechanisms and a majority of them inhibit the nuclear factor kappa-beta (NF-κB) signaling pathway. In this study, we examined the effects of the naturally occurring PTs on IκB kinase-β (IKKβ), which has great scientific relevance in the NF-κB signaling pathway. On virtual screening, 109 PTs were screened through the PASS (prediction of activity spectra of substances) software for prediction of NF-κB inhibitory activity followed by docking on the NEMO/IKKβ association complex (PDB: 3BRV) and testing for compliance with the softened Lipinski’s Rule of Five using Schrodinger (LLC, New York, USA). Out of the projected 45 druggable PTs, Corosolic Acid (CA), Asiatic Acid (AA) and Ursolic Acid (UA) were assayed for IKKβ kinase activity in the cell free medium. The UA exhibited a potent IKKβ inhibitory effect on the hotspot kinase assay with IC50 of 69 μM. Whereas, CA at 50 μM concentration markedly reduced the NF-κB luciferase activity and phospho-IKKβ protein expressions. The PTs tested, attenuated the expression of the NF-κB cascade proteins in the LPS-stimulated RAW 264.7 cells, prevented the phosphorylation of the IKKα/β and blocked the activation of the Interferon-gamma (IFN-γ). The results suggest that the IKKβ inhibition is the major mechanism of the PTs-induced NF-κB inhibition. PASS predictions along with in-silico docking against the NEMO/IKKβ can be successfully applied in the selection of the prospective NF-κB inhibitory downregulators of IKKβ phosphorylation.
Highlights
Nuclear factor kappa B (NF-κB) is a family of ubiquitous transcription factors comprising five related elements, namely p50, p52, RelA, RelB and c-Rel [1,2,3]
The virtual screening protocol used in this study is based on the sequential application of filters to explore the selective IKKβ mediated NF-κB inhibitory Pentacyclic Triterpenoids (PTs)
The workflow was as shown in S1 Fig. The virtual screening resulted in the establishment of a compound library containing 109 prospective NF-κB inhibitory PTs
Summary
Nuclear factor kappa B (NF-κB) is a family of ubiquitous transcription factors comprising five related elements, namely p50, p52, RelA, RelB and c-Rel [1,2,3]. The upstream stimuli, like lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α) and Interleukin-1 (IL-1) activate the IκB kinase (IKK) complex, consisting of catalytic IKKα and IKKβ subunits along with the regulatory subunit IKKγ termed NEMO (NF-κB Essential Modulator) [10,11]. In both pathways; classical and alternative IKK activation is a common regulatory step initiating the NF-κB signaling. The transcriptional activity of NF-κB induces the expression of IκBα gene and generates IκBα, which sequesters the NF-κB subunits and terminates the transcriptional activity of NF-κB [15]
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