International Journal of Biological Macromolecules 96 (2017) 200–213 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Identification of natural allosteric inhibitor for Akt1 protein through computational approaches and in vitro evaluation T. Pragna Lakshmi a , Amit Kumar a , Veena Vijaykumar b , Sakthivel Natarajan b , Ramadas Krishna a,∗ a Centre for Bioinformatics, Pondicherry University, Puducherry 605014, India b Department of Biotechnology, Pondicherry University, Puducherry 605014, India a r t i c l e i n f o Article history: Received 24 August 2016 Received in revised form 2 December 2016 Accepted 4 December 2016 Available online 9 December 2016 Keywords: Breast cancer cell line Cloning Protein expression and purification Isothermal titration calorimetry a b s t r a c t 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--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 (K d ) of 0.246 M. With these computational, biological and thermodynamic studies, we suggest Q7G as a lead molecule and propose for its further optimization. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Akt or PKB is a key effector of PI3K/Akt/mTOR pathway that plays pivotal role in cellular metabolism, growth, proliferation, differentiation and survival [1]. Currently, there are three known isoforms of Akt: Akt1, 2 and 3 or PKB-,  and  [2,3]. Activa- tion of Akt is dependent on stimulation of receptor tyrosine kinase (RTK) or G-protein coupled receptor (GPCR) which triggers phos- phatidylinositol 3-kinase (PI3K) to generate phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) in the cell membrane. This drives the Abbreviations: PKB, protein kinase B; CPH, common pharmacophore hypothe- sis; RMSE, root mean-square error; PLS, partial least-squares; QSAR, quantitative structure-activity relationship; TCM, traditional Chinese medicine; HTVS, high- throughput virtual screening; SP, standard precision; XP, extra precision; DFT, density functional theory; HOMO, higher occupied frontier orbitals; LUMO, lower unoccupied frontier orbitals; MDS, molecular dynamic simulations. ∗ Corresponding author. E-mail addresses: pragnapcu@gmail.com (T. Pragna Lakshmi), amit.kumar10nov@gmail.com (A. Kumar), btveenavijaykumar@gmail.com (V. Vijaykumar), puns2005@gmail.com (S. Natarajan), krishstrucbio@gmail.com (R. Krishna). recruitment of Akt and other pleckstrin homology (PH) domain containing proteins such as phosphoinositide-dependent kinase- 1 (PDK1) to the inner leaflet of plasma membrane, where Akt comes in close proximity to PDK1 and gets phosphorylated at Thr308 of catalytic domain. Akt is fully activated when it is phos- phorylated at Ser473 either by mammalian target of rapamycin complex2 (mTORC2) or by DNA-dependent protein kinase (DNA- PK). Activated Akt stimulates several downstream substrates which finally lead to inhibition of apoptosis, promotion of cellular growth, survival, glucose metabolism and angiogenesis [4–9]. In various cancers such as breast and prostate, Akt is constantly active or over-expressed due to its own mutations or aberrant activity of upstream proteins of the pathway [10]. Inhibition of Akt could reverse its anti-apoptotic processes and thereby regulate cellu- lar growth, proliferation and survival. Besides, hyper activation of Akt often increases resistance to chemotherapy or radiotherapy. Akt inhibitors have been shown to attenuate this chemotherapeu- tic resistance when they were administrated along with standard chemotherapy [11–13]. Akt structure comprises of an amino (N)-terminal regula- tory PH domain, 42-residue length connecting linker, catalytic http://dx.doi.org/10.1016/j.ijbiomac.2016.12.025 0141-8130/© 2016 Elsevier B.V. All rights reserved.