title: Small molecules preventing GAPDH aggregation are therapeutically applicable in cell and rat models of oxidative stress.
abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most abundant targets of the oxidative stress. Oxidation of the enzyme causes its inactivation and the formation of intermolecular disulfide bonds, and leads to the accumulation of GAPDH aggregates and ultimately to cell death. The aim of this work was to reveal the ability of chemicals to break the described above pathologic linkage by inhibiting GAPDH aggregation. Using the model of oxidative stress based on SK-N-SH human neuroblastoma cells treated with hydrogen peroxide, we found that lentivirus-mediated down- or up-regulation of GAPDH content caused inhibition or enhancement of the protein aggregation and respectively reduced or increased the level of cell death. To reveal substances that are able to inhibit GAPDH aggregation, we developed a special assay based on dot ultrafiltration using the collection of small molecules of plant origin. In the first round of screening, five compounds were found to possess anti-aggregation activity as established by ultrafiltration and dynamic light scattering; some of the substances efficiently inhibited GAPDH aggregation in nanomolar concentrations. The ability of the compounds to bind GAPDH molecules was proved by the drug affinity responsive target stability assay, molecular docking and differential scanning calorimetry. Results of experiments with SK-N-SH human neuroblastoma treated with hydrogen peroxide show that two substances, RX409 and RX426, lowered the degree of GAPDH aggregation and reduced cell death by 30%. Oxidative injury was emulated in vivo by injecting of malonic acid into the rat brain, and we showed that the treatment with RX409 or RX426 inhibited GAPDH-mediated aggregation in the brain, reduced areas of the injury as proved by magnetic resonance imaging, and augmented the behavioral status of the rats as established by the "beam walking" test. In conclusion, the data show that two GAPDH binders could be therapeutically relevant in the treatment of injuries stemming from hard oxidative stress.
authors.affiliation: Institute of Cytology of Russian Academy of Sciences, Tikhoretsky pr., 4, 194064 St. Petersburg, Russia. Electronic address: vl.lazarev@gmail.com.
authors.forename: Vladimir F
authors.initials: VF
authors.lastname: Lazarev
authors.affiliation: Institute of Cytology of Russian Academy of Sciences, Tikhoretsky pr., 4, 194064 St. Petersburg, Russia.
authors.forename: Alina D
authors.initials: AD
authors.lastname: Nikotina
authors.affiliation: Belozersky Institute of Physico-Chemical Biology of Moscow State University, 119992 Moscow, Russia.
authors.forename: Pavel I
authors.initials: PI
authors.lastname: Semenyuk
authors.affiliation: Belozersky Institute of Physico-Chemical Biology of Moscow State University, 119992 Moscow, Russia.
authors.forename: Diana B
authors.initials: DB
authors.lastname: Evstafyeva
authors.affiliation: Institute of Cytology of Russian Academy of Sciences, Tikhoretsky pr., 4, 194064 St. Petersburg, Russia.
authors.forename: Elena R
authors.initials: ER
authors.lastname: Mikhaylova
authors.affiliation: Belozersky Institute of Physico-Chemical Biology of Moscow State University, 119992 Moscow, Russia.
authors.forename: Vladimir I
authors.initials: VI
authors.lastname: Muronetz
authors.affiliation: Institute of Cytology of Russian Academy of Sciences, Tikhoretsky pr., 4, 194064 St. Petersburg, Russia.
authors.forename: Maxim A
authors.initials: MA
authors.lastname: Shevtsov
authors.affiliation: Institute of Cytology of Russian Academy of Sciences, Tikhoretsky pr., 4, 194064 St. Petersburg, Russia.
authors.forename: Anastasia V
authors.initials: AV
authors.lastname: Tolkacheva
authors.affiliation: Institute of Macromolecular Compounds Russian Academy of Sciences, 199004 St. Petersburg, Russia.
authors.forename: Anatoly V
authors.initials: AV
authors.lastname: Dobrodumov
authors.affiliation: Komarov Botanical Institute Russian Academy of Sciences, 197376 St. Petersburg, Russia.
authors.forename: Alexey L
authors.initials: AL
authors.lastname: Shavarda
authors.affiliation: Institute of Cytology of Russian Academy of Sciences, Tikhoretsky pr., 4, 194064 St. Petersburg, Russia.
authors.forename: Irina V
authors.initials: IV
authors.lastname: Guzhova
authors.affiliation: Institute of Cytology of Russian Academy of Sciences, Tikhoretsky pr., 4, 194064 St. Petersburg, Russia.
authors.forename: Boris A
authors.initials: BA
authors.lastname: Margulis
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002777:Cholestanols / Q000008:administration & dosage*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019791:Guanidine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D062105:Molecular Docking Simulation; D009447:Neuroblastoma / Q000188:drug therapy / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects*; D066263:Protein Aggregation, Pathological / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051381:Rats; D054852:Small Molecule Libraries / Q000008:administration & dosage* / Q000737:chemistry
chemical_list: D002777:Cholestanols; C000612084:RX409 compound; C000612085:RX426 compound; D054852:Small Molecule Libraries; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019791:Guanidine
keywords: Aggregates; Drug; Glyceraldehyde-3-phosphate dehydrogenase; Oxidative stress; Small molecule