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Identification of natural allosteric inhibitor for Akt1 protein through computational approaches and in vitro

T Pragna Lakshmi1, Amit Kumar1, Veena Vijaykumar2

  • 1Centre for Bioinformatics, Pondicherry University, Puducherry 605014, India.

International Journal of Biological Macromolecules
|December 14, 2016
PubMed

Insights

Quercetin-7-O-β-d-glucopyranoside (Q7G) shows potential as a natural allosteric inhibitor for Akt1, a key kinase in cancer. Q7G effectively inhibits breast cancer cell growth by stabilizing Akt1 in an inactive state.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • Akt, a serine/threonine kinase, is frequently hyperactivated in breast and prostate cancers, correlating with poor patient prognosis.
  • Allosteric inhibitors offer a therapeutic strategy by stabilizing kinases in an inactive conformation, thereby regulating aberrant activity.

Purpose of the Study:

  • To identify novel natural allosteric inhibitors of Akt1 using computational and experimental approaches.
  • To evaluate the potential of Quercetin-7-O-β-d-glucopyranoside (Q7G) as an Akt1 inhibitor.

Main Methods:

  • Generation of a seven-point pharmacophore model for Akt1.
  • Screening of a natural compound library against the pharmacophore model.
  • Molecular dynamics simulations to assess binding stability and free energy.
  • In vitro assays to evaluate Q7G's effect on breast cancer cell lines (MDA MB-231).
  • Analysis of apoptosis markers (Bcl-2, caspase-3, PARP) and Akt phosphorylation (p-Akt).
  • Thermodynamic analysis of Akt1-Q7G interaction using dissociation constant (Kd).

Main Results:

  • Q7G demonstrated strong binding affinity to Akt1, stabilizing it in an inactive conformation through key allosteric residue interactions.
  • Q7G induced dose-dependent inhibition of MDA MB-231 breast cancer cells, causing G1 and sub-G phase arrest.
  • Q7G treatment led to decreased levels of the anti-apoptotic protein Bcl-2 and increased levels of cleaved caspase-3 and PARP, indicating apoptosis induction.
  • Down-regulation of p-Akt (Ser473) was observed, consistent with Akt1 inhibition.
  • The dissociation constant (Kd) for the Akt1-Q7G interaction was determined to be 0.246 μM.

Conclusions:

  • Q7G acts as a potent natural allosteric inhibitor of Akt1, demonstrating significant anti-cancer effects in vitro.
  • Q7G stabilizes Akt1 in an inactive conformation, leading to cell cycle arrest and apoptosis in breast cancer cells.
  • Q7G represents a promising lead molecule for further optimization and development as a targeted cancer therapeutic.
Keywords:
Breast cancer cell lineCloningIsothermal titration calorimetryProtein expression and purification

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