Synthesis and characterization of a novel class of reducing agents that are highly neuroprotective for retinal ganglion cells

Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, 600 Highland Avenue, Madison, WI 52792, USA.
Experimental Eye Research (Impact Factor: 2.71). 12/2006; 83(5):1252-9. DOI: 10.1016/j.exer.2006.07.002
Source: PubMed


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.

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Available from: Christopher R Schlieve, May 01, 2014
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    • "PB1 and PB2 were designed to have low reactivity in the extracellular compartment, high rates of transmembrane diffusion, and a side-group that can be cleaved by intracellular enzymes, resulting in an intracellular accumulation of PB1 or PB2. PB1 and PB2 are neuroprotective in vitro in axotomized primary retinal ganglion cells [27] and a neuronal cell line where the mitochondrial electron transport chain components are inhibited [30]. They are also neuroprotective in vivo in rat optic nerve crush and ocular hypertension models [31]. "
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    ABSTRACT: Exposure to radiation can damage endothelial cells in the irradiated area via the production of reactive oxygen species. We synthesized phosphine-borane complexes that reduce disulfide bonds and had previously been shown to interfere with redox-mediated signaling of cell death. We hypothesized that this class of drugs could interfere with the downstream effects of oxidative stress after irradiation and rescue endothelial cells from radiation damage. Cultured bovine aortic endothelial cells were plated for clonogenic assay prior to exposure to varying doses of irradiation from a (137)Cs irradiator and treated with various concentrations of bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) at different time points. The clone-forming ability of the irradiated cells was assessed seven days after irradiation. We compared the radioprotective effects of PB1 with the aminothiol radioprotectant WR1065 and known superoxide scavengers. PB1 significantly protected bovine aortic endothelial cells from radiation damage, particularly when treated both before and after radiation. The radioprotection with 1µM PB1 corresponded to a dose-reduction factor of 1.24. Radioprotection by PB1 was comparable to the aminothiol WR1065, but was significantly less toxic and required much lower concentrations of drug (1µM vs. 4mM, respectively). Superoxide scavengers were not radioprotective in this paradigm, indicating the mechanisms for both loss of clonogenicity and PB1 radioprotection are independent of superoxide signaling. These data demonstrate that PB1 is an effective redox-active radioprotectant for endothelial cells in vitro, and is radioprotective at a concentration approximately 4 orders of magnitude lower than the aminothiol WR1065 with less toxicity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
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    • "Human glaucomatous retinas contain high levels of the lipid peroxidation indicator 4-hydroxy-2-nonenal (HNE), which leads to protein modification induced by superoxide (Tezel et al. 2010). Administration of pegylated superoxide dismutase-1 (SOD), which catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide (H 2 O 2 ), attenuates RGC death (Kanamori et al. 2010, Schlieve et al. 2006) supporting the idea that interfering with superoxide generation might be beneficial. However, the translation of a protein-based therapy that requires intracellular delivery is considerably more challenging than a small molecule approach. "
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    ABSTRACT: J. Neurochem. (2011) 118, 1075–1086. The reactive oxygen species (ROS) superoxide has been recognized as a critical signal triggering retinal ganglion cell (RGC) death after axonal injury. Although the downstream targets of superoxide are unknown, chemical reduction of oxidized sulfhydryls has been shown to be neuroprotective for injured RGCs. On the basis of this, we developed novel phosphine-borane complex compounds that are cell permeable and highly stable. Here, we report that our lead compound, bis (3-propionic acid methyl ester) phenylphosphine borane complex 1 (PB1) promotes RGC survival in rat models of optic nerve axotomy and in experimental glaucoma. PB1-mediated RGC neuroprotection did not correlate with inhibition of stress-activated protein kinase signaling, including apoptosis stimulating kinase 1 (ASK1), c-jun NH2-terminal kinase (JNK) or p38. Instead, PB1 led to a striking increase in retinal BDNF levels and downstream activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Pharmacological inhibition of ERK1/2 entirely blocked RGC neuroprotection induced by PB1. We conclude that PB1 protects damaged RGCs through activation of pro-survival signals. These data support a potential cross-talk between redox homeostasis and neurotrophin-related pathways leading to RGC survival after axonal injury.
    Preview · Article · Jul 2011 · Journal of Neurochemistry

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