Securidaca–saponins are natural inhibitors of AKT, MCL-1, and BCL2L1 in cervical cancer cells

Titus Chukwuemeka Obasi,1 Cornelia Braicu,2 Bogdan Cezar Iacob,1 Ede Bodoki,1 Ancuta Jurj,2 Lajos Raduly,2 Ilioara Oniga,3 Ioana Berindan-Neagoe,2,4,5 Radu Oprean1 1Department of Analytical Chemistry and Instrumental Analysis, Faculty of Pharmacy, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania; 2Research Center for Functional Genomics, Biomedicine and Translational Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania; 3Department of Pharmacognosy, Faculty of Pharmacy, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania; 4MEDFUTURE – Research Center for Advanced Medicine, University of Medicine and Pharmacy Iuliu-Hatieganu, Cluj-Napoca, Romania; 5Department of Functional Genomics and Experimental Pathology, The Oncology Institute “Prof. Dr. Ion Chiricuta”, Cluj-Napoca, Romania Introduction: Scientific research is beginning to prove the connection between claims by African traditional medicine and the natural chemical specifics contained in medicinal plant Securidaca longipedunculata. Our previous studies showed that two natural saponin fractions (4A3 and 4A4) identified in the plant as triterpenoid glycosides are capable of activating apoptosis on cervical tumor cell lines. Considering this and some critical roles of human papillomavirus (HPV) E6 oncogene on cervical cells, by promoting carcinogenesis and cell survival, it became necessary to investigate the possible pathways for apoptosis transmission.Methods: Tests conducted on relevant cervical tumor cell lines such as Caski and Bu25TK included the following: MTT assay; scratch assay (to determine cell migration/invasion); fluorescence microscopy with Annexin V–fluorescein isothiocyanate, muscle progenitor cell) and propidium iodide staining; and finally reverse transcriptase quantitative PCR (RT-qPCR) for gene analysis.Results: Reduced cell proliferation was observed due to activities of 4A3 and 4A4 fractions, with half-maximal inhibitory concentration (IC50) of 7.03 and 16.39 μg/mL, respectively, on Caski cell line. A significant reduction in cell migration occurred within 48 and 72 hours, respectively, for Caski and Bu25TK cell lines. Late apoptosis was activated by 4A3, staining both Annexin V and PI, in contrast to 4A4’s early apoptosis. RT-qPCR data revealed a fold change (FC) inhibition of antiapoptotic proteins such as MCL-1 and BCL2L1, with diminished level of AKT-3, VEGFA, MALAT1, etc. The expression of p53, proapoptotic BAD, and caspase-8 was nonsignificant.Conclusion: The low expression of AKT-3 and antiapoptotic proteins (MCL-1 and BCL2L1), as well as VEGFA, could simply be an indication for possible suppression of cell survival mechanisms via multiple channels. We therefore conclude that 4A3 and 4A4 fractions mediate activity via the inhibition of phosphatidylinositol-3-OH kinase (PI3K)-AKT/mTOR/NF-kB-dependent antiapoptotic stimuli. Further studies are ongoing to reveal the chemical structures and compositions of these two fractions. Keywords: early apoptosis, RT-qPCR gene analysis, AKT-3, MCL-1 and BCL2L1 inhibition, triterpenoid saponin

Beta decay of Ga-62

Journals published by the American Physical Society can be found at http://publish.aps.org/We report a study of the beta decay of Ga-62, whose dominant branch is a superallowed 0(+)-->0(+) transition to the ground state of Zn-62. We find the total half-life to be 115.84+/-0.25 ms. This is the first time that the Ga-62 half-life has been measured with a purified source. We find that (0.120+/-0.021)% of the beta decays are followed by gamma cascades that pass through the Zn-62 2(+) first excited state at 0.954 MeV. The branching ratio to the first-excited 0(+) state in Zn-62 at 2.33 MeV is <0.043%. We conclude that the branching ratio for the superallowed transition is 99.85(-0.15)(+0.05)%

Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating residues. This study has outlined mechanisms by which inhibitor binding could modulate resilience and efficiency of allosteric interactions in the kinase structures, while preserving structural topology required for catalytic activity and regulation

Interrogating Regulatory Mechanisms in Signaling Proteins by Allosteric Inhibitors and Activators: A Dynamic View Through the Lens of Residue Interaction Networks

Computational studies of allosteric interactions have witnessed a recent renaissance fueled by the growing interest in modeling of the complex molecular assemblies and biological networks. Allosteric interactions in protein structures allow for molecular communication in signal transduction networks. In this chapter, we discuss recent developments in understanding of allosteric mechanisms and interactions of protein systems, particularly in the context of structural, functional, and computational studies of allosteric inhibitors and activators. Computational and experimental approaches and advances in understanding allosteric regulatory mechanisms are reviewed to provide a systematic and critical view of the current progress in the development of allosteric modulators and highlight most challenging questions in the field. The abundance and diversity of genetic, structural, and biochemical data underlies the complexity of mechanisms by which targeted and personalized drugs can combat mutational profiles in protein kinases. Structural and computational studies of protein kinases have generated in recent decade significant insights that allowed leveraging knowledge about conformational diversity and allosteric regulation of protein kinases in the design and discovery of novel kinase drugs. We discuss recent developments in understanding multilayered allosteric regulatory machinery of protein kinases and provide a systematic view of the current state in understanding molecular basis of allostery mediated by kinase inhibitors and activators. In conclusion, we highlight the current status and future prospects of computational biology approaches in bridging the basic science of protein kinases with the discovery of anticancer therapies.https://digitalcommons.chapman.edu/scs_books/1049/thumbnail.jp