title: Identification of natural allosteric inhibitor for Akt1 protein through computational approaches and in vitro evaluation.
abstract: Akt, a serine/threonine protein kinase, is often hyper activated in breast and prostate cancers, but with poor prognosis. Allosteric inhibitors regulate aberrant kinase activity by stabilizing the protein in inactive conformation. Several natural compounds have been reported as inhibitors for kinases. In this study, to identify potential natural allosteric inhibitor for Akt1, we generated a seven-point pharmacophore model and screened it through natural compound library. Quercetin-7-O-β-d-glucopyranoside or Q7G was found to be the best among selected molecules based on its hydrogen bond occupancy with key allosteric residues, persistent polar contacts and salt bridges that stabilize Akt1 in inactive conformation and minimum binding free energy during molecular dynamics simulation. Q7G induced dose-dependent inhibition of breast cancer cells (MDA MB-231) and arrested them in G1 and sub-G phase. This was associated with down-regulation of anti-apoptotic protein Bcl-2, up-regulation of cleaved caspase-3 and PARP. Expression of p-Akt (Ser473) was also down-regulated which might be due to Akt1 inhibition in inactive conformation. We further confirmed the Akt1 and Q7G interaction which was observed to have a dissociation constant (Kd) of 0.246μM. With these computational, biological and thermodynamic studies, we suggest Q7G as a lead molecule and propose for its further optimization.
authors.affiliation: Centre for Bioinformatics, Pondicherry University, Puducherry 605014, India. Electronic address: pragnapcu@gmail.com.
authors.forename: T
authors.initials: T
authors.lastname: Pragna Lakshmi
authors.affiliation: Centre for Bioinformatics, Pondicherry University, Puducherry 605014, India. Electronic address: amit.kumar10nov@gmail.com.
authors.forename: Amit
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Biotechnology, Pondicherry University, Puducherry 605014, India. Electronic address: btveenavijaykumar@gmail.com.
authors.forename: Veena
authors.initials: V
authors.lastname: Vijaykumar
authors.affiliation: Department of Biotechnology, Pondicherry University, Puducherry 605014, India. Electronic address: puns2005@gmail.com.
authors.forename: Sakthivel
authors.initials: S
authors.lastname: Natarajan
authors.affiliation: Centre for Bioinformatics, Pondicherry University, Puducherry 605014, India. Electronic address: krishstrucbio@gmail.com.
authors.forename: Ramadas
authors.initials: R
authors.lastname: Krishna
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D017209:Apoptosis / Q000187:drug effects; D001688:Biological Products / Q000737:chemistry / Q000494:pharmacology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D006801:Humans; D056004:Molecular Dynamics Simulation; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry* / Q000378:metabolism*; D021281:Quantitative Structure-Activity Relationship; D013816:Thermodynamics; D014584:User-Computer Interface
chemical_list: D001688:Biological Products; D051057:Proto-Oncogene Proteins c-akt
keywords: Breast cancer cell line; Cloning; Isothermal titration calorimetry; Protein expression and purification