title: Synthesis and characterization of a novel class of reducing agents that are highly neuroprotective for retinal ganglion cells.
abstract: Retinal ganglion cells (RGCs) undergo apoptosis after axonal injury, in part regulated by an intracellular superoxide anion burst, for which the target(s) are unknown. Shifting the RGC redox state towards reduction and preventing sulfhydryl oxidation is neuroprotective in vitro and in vivo, implying that one or more sulfhydryls on one or more critical proteins may be involved. We synthesized novel borane-protected analogues of the reductant tris(2-carboxyethyl)phosphine (TCEP) with the intent of increasing cell permeability and improving chemical stability, and tested their ability to increase RGC survival in vitro. Retinal ganglion cells of postnatal day 2-4 Long-Evans rats were retrogradely labeled with 4',6-diamidino-2-phenylindole (DAPI). At postnatal days 11-13 the animals were sacrificed, the retinas enzymatically dissociated and plated on poly-L-lysine-coated 96-well flat-bottomed tissue culture plates for 72 h in Neurobasal-A, B27 supplement lacking antioxidants, and TCEP, bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1), (3-propionic acid methyl ester)diphenylphosphine borane complex (PB2), or three commercially available phosphines. Viable DAPI-positive RGCs were identified by calcein-AM staining. At 72 h, PB1 was effective at rescuing acutely axotomized RGCs at concentrations from 1 nM to 100 microM. RGC survival with 1 nM PB1 was 174+/-12% of control (p=0.002). Another compound, PB2, rescued RGCs at 10 pM (177+/-24%; p=0.006) and 10 nM (251+/-34%; p=0.004) at 72 h. A PAMPA assay demonstrated that PB1 and PB2 were substantially more permeable than TCEP. These data demonstrate that modified reductants are effective RGC neuroprotectants at picomolar-nanomolar concentrations. We propose that these novel molecules may act by inhibiting the sulfhydryl oxidation effect of an intracellular superoxide burst.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, 600 Highland Avenue, Madison, WI 52792, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Schlieve
authors.forename: Annie
authors.initials: A
authors.lastname: Tam
authors.forename: Bradley L
authors.initials: BL
authors.lastname: Nilsson
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Lieven
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Raines
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Levin
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001880:Boranes / Q000737:chemistry / Q000494:pharmacology; D002452:Cell Count; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D007202:Indicators and Reagents / Q000737:chemistry; D018696:Neuroprotective Agents / Q000737:chemistry / Q000494:pharmacology*; D009900:Optic Nerve; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D020318:Rats, Long-Evans; D019163:Reducing Agents / Q000737:chemistry / Q000494:pharmacology; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
chemical_list: D001880:Boranes; D007202:Indicators and Reagents; D018696:Neuroprotective Agents; D010720:Phosphines; D019163:Reducing Agents; C515674:bis(3-propionic acid methyl ester)diphenylphosphine borane complex; C515673:bis(3-propionic acid methyl ester)phenylphosphine borane complex; C080938:tris(2-carboxyethyl)phosphine