Targeting DNA Repair through Podophyllotoxin and Rutin Formulation in Hematopoietic Radioprotection: An in Silico, in Vitro, and in Vivo Study.

Pankaj Chaudhary,Nitya N Srivastava,M H Yashavarddhan,Manju L Gupta,Mrutyunjay Suar,Sandeep K Shukla,Jayadev Joshi

doi:10.3389/fphar.2017.00750

Abstract

Drug discovery field has tremendously progressed during last few decades, however, an effective radiation countermeasure agent for the safe administration to the victims of radiation exposure is still unavailable. This multi-model study is aimed at elucidating the mechanistic aspects of a novel podophyllotoxin and rutin combination (henceforth referred as G-003M) in the hematopoietic radioprotection and its involvement in the DNA damage and repair signaling pathways. Using in silico study, we identified the binding sites and structural components of G-003M and validated in vitro. We further studied various in vivo endpoints related to the DNA repair and cell death pathways in mice pre-administered with G-003M, irradiated and subsequently euthanized to collect blood and bone marrow cells. In silico study showed the binding of podophyllotoxin to β-tubulin and presence of a functional hydroxyl group in the rutin, suggested their involvement in G2/M arrest and the free radical scavenging respectively. This experimentation was further validated through in vitro studies. In vivo mice studies confirmed that G-003M pre-administration attenuated DNA damage and enhanced repair after whole body exposure. We further noticed a decrease in the levels of γH2AX, p53BP1, and ATM kinase and an increase in the levels of DNA pk, Ku 80, Ligase IV, Mre 11, Rad 50 and NBS 1 in the blood and bone marrow cells of the G-003M pre-administered and irradiated mice. We noticed an overall increase in the pro-survival factors in the G-003M pre-treated and irradiated groups establishing the radioprotective efficacy of this formulation. The lead obtained from this study will certainly help in developing this formulation as a safe and effective radioprotector which could be used for humans against any planned or emergency exposure of radiation.

Highlights

United States Food and Drug Administration (FDA) has approved only a few radiation countermeasure agents with limited applications suitable for radiation victims
We have investigated the mode of action of G-003M by analyzing the property of individual compounds, i.e., podophyllotoxin and rutin with in silico, in vitro and in vivo methods
Properties of Podophyllotoxin and Rutin An in silico study was designed using PubChem bioactivity data base to investigate the off-targets of podophyllotoxin

Summary

Introduction

United States Food and Drug Administration (FDA) has approved only a few radiation countermeasure agents with limited applications suitable for radiation victims. FDA approved safe radioprotector is highly desirable by armed forces and patients undergoing radiotherapy. Podophyllotoxin (Figure 1) is a lignan, that directly targets DNA topoisomerase-2α (Zhang et al, 1992; Kamal et al, 2005), tubulin alpha-4A chain (Screpanti et al, 2010) and tubulinβ chain (Wolff et al, 1991) confirming its involvement in the mitotic arrest and rejoining of DNA strand breaks (Wolff et al, 1991; Kamal et al, 2005). Podophyllotoxin is further involved in the enzymatic activation of Cytochrome P4502C19 and P450-3A4 important in NADPH-dependent electron transport pathway (Preissner et al, 2010). Rutin targets Aldo-k-keto-reductase-1-member-C3 (Imming et al, 2006) and activates enzymes Cytochrome-P450-2C8, P450-2C9, P4502D6 and P450-3A4, which subsequently regulates metabolic processes, cell proliferation and death

Methods

Results

Conclusion