Folic Acid Exerts Post-Ischemic Neuroprotection In Vitro Through HIF-1α Stabilization

Abstract

The constant failure of single-target drug therapies for ischemic stroke necessitates the development of novel pleiotropic pharmacological treatment approaches, to effectively combat the aftermath of this devastating disorder. The major objective of our study involves a multi-target drug repurposing strategy to stabilize hypoxia-inducible factor-1 α (HIF-1α) via a structure-based screening approach to simultaneously inhibit its regulatory proteins, PHD2, FIH, and pVHL. Out of 1424 Food and Drug Administration (FDA)-approved drugs that were screened, folic acid (FA) emerged as the top hit and its binding potential to PHD2, FIH, and pVHL was further verified by re-docking, molecular dynamics (MD) simulation and by Drug Affinity Responsive Target Stability (DARTS) assay. HIF-1α stabilization by FA was demonstrated by the nuclear translocation and increased green fluorescence emission of HIF-1α using HIF1α-GFPSpark tag vector. Further, FA treatment enhanced the cell survival following oxygen glucose deprivation and its neuroprotective mechanism was elucidated by measuring the expression of BAX, NFE2L2, VEGF, and EPO genes in a time-dependent manner (5 and 11 h following FA treatment). VEGF and EPO expressions were significantly increased by 5.41- and 1.35-folds, respectively, whereas BAX expression reduced by 4-fold at 11 h post-FA treatment. NFE2L2 expression was elevated (1.65-fold) at 5 h with no major difference at 11 h post-FA treatment. The chicken chorioallantoic membrane (CAM) assay demonstrated the pro-angiogenic potential of FA as evidenced by an increased blood vessel density and branching. The present study elucidates for the first time that the post-ischemic neuroprotection exerted by FA may be attributed to its HIF-1α stabilization and pro-angiogenic properties.

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Abbreviations

ANOVA:

Analysis of variance

ARE:

Anti-oxidant response element

ATP:

Adenosine triphosphate

BAX:

BCL2 associated x, apoptosis regulator

CAM:

Chick chorioallantoic membrane

DARTS:

Drug affinity responsive target stability

DMEM:

Dulbecco’s modified Eagle’s medium

DMOG:

Dimethyloxaloylglycine

EPO:

Erythropoietin

FA:

Folic acid

FBS:

Fetal bovine serum

FDA:

Food and drug administration

FIH:

Factor inhibiting HIF

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GFP:

Green fluorescent protein

HIF-1α:

Hypoxia-inducible factor-1 alpha

MALDI-TOF MS:

Matrix assisted laser desorption/ionization - time of flight mass spectrometry

MD:

Molecular dynamics

MM/GBSA:

Molecular mechanics/generalized-Born surface area

MTHFR:

Methylene tetrahydrofolate reductase

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NFE2L2:

Nuclear factor, erythroid 2 like 2

NRF2:

Nuclear factor erythroid 2–related factor 2

OGD:

Oxygen glucose deprivation

OPLS-AA:

Optimized potentials for liquid simulations-all atom

PBS:

Phosphate-buffered saline

PDB:

Protein data bank

PHD2:

HIF prolyl hydroxylase 2

PMSF:

Phenylmethyl sulfonyl fluoride

pVHL:

Von Hippel−Lindau protein

qPCR:

Quantitative PCR

Rg :

Radius of gyration

RMSD:

Root-mean-square deviation

RMSF:

Root-mean-square fluctuation

ROS:

Reactive oxygen species

RPMI:

Roswell Park memorial institute

RT-PCR:

Real-time-polymerase chain reaction

SDS:

Sodium dodecyl sulphate

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SEM:

Standard error of the mean

VEGF:

Vascular endothelial growth factor

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Acknowledgements

The authors would like to thank Dr. Fayaz S. M. (Manipal Institute of Technology, India) for the technical support on molecular docking and MD simulation studies.

Funding

This research was supported by the Department of Biotechnology, Government of India, New Delhi, India (Grant numbers BT/BI/25/001/2006 and BT/PR13909/MED/30/305/2010). The computational portion of the study was carried out in the “Bioinformatics Infrastructure Facility for Biology Teaching through Bioinformatics (BIF-BTBI)” (Grant number BT/BI/25/001/2006 dated 25/03/2011), which was financially supported by the Department of Biotechnology, Government of India.

Author information

Affiliations

Authors

Contributions

CDK and RGK planned the experiments; CDK performed the experiments and analyzed the data; SN and CDK wrote the paper; and RGK revised the manuscript critically and approved the final version to be submitted.

Corresponding author

Correspondence to G. K. Rajanikant.

Electronic supplementary material

ESM 1
figure10

Animation showing FA bound to PHD2?at the active site during the course of MD simulation. (GIF 646 kb)

ESM 2
figure11

The diagram shows the minor (white), moderate (light grey) or serious (dark grey) interference of FA with other drugs. The data were obtained using Drug interaction checker and graphically presented using cytoscape 3.5.0.. (GIF 18.8 kb)

12035_2018_982_MOESM1_ESM.mp4

Animation showing FA bound to FIH at the active site during the course of MD simulation. (MP4 4089 kb)

12035_2018_982_MOESM2_ESM.mp4

Animation showing FA bound to pVHL at the active site during the course of MD simulation. (MP4 7459 kb)

12035_2018_982_MOESM3_ESM.mp4

The graph shows the fold changes in HIF1-a expression between control, OGD and FA treated OGD groups. Error bars indicate SEM (OGD vs treatment group, * p??<??0.05). (MP4 3048 kb)

ESM 3

Animation showing FA bound to FIH at the active site during the course of MD simulation. (MP4 4089 kb)

ESM 4

Animation showing FA bound to pVHL at the active site during the course of MD simulation. (MP4 7459 kb)

ESM 5

The graph shows the fold changes in HIF1-a expression between control, OGD and FA treated OGD groups. Error bars indicate SEM (OGD vs treatment group, * p??<??0.05). (MP4 3048 kb)

High resolution image (TIFF 679 kb)

High resolution image (TIFF 665 kb)

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Davis, C.K., Nampoothiri, S.S. & Rajanikant, G.K. Folic Acid Exerts Post-Ischemic Neuroprotection In Vitro Through HIF-1α Stabilization. Mol Neurobiol 55, 8328–8345 (2018). https://doi.org/10.1007/s12035-018-0982-3

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Keywords

  • Molecular docking
  • Molecular dynamics simulation
  • Folic acid
  • Drug repurposing
  • Neuroprotection
  • Hypoxia-inducible factor 1