JM-20, a novel benzodiazepine–dihydropyridine hybrid molecule, protects mitochondria and prevents ischemic insult-mediated neural cell death in vitro

Abstract

The ischemic stroke cascade is composed of several pathophysiological events, providing multiple targets for pharmacological intervention. JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine) is a novel hybrid molecule, in which a benzodiazepine portion is covalently linked to a dihydropyridine ring, forming a new chemical entity with potential multisite neuroprotective activity. In the present study, JM-20 prevented PC-12 cell death induced either by glutamate, hydrogen peroxide or KCN-mediated chemical hypoxia. This molecule also protected cerebellar granule neurons from glutamate or glutamate plus pentylenetetrazole-induced damage at very low micromolar concentrations. In rat liver mitochondria, JM-20, at low micromolar concentrations, prevented the Ca2+-induced mitochondrial permeability transition, as assessed by mitochondrial swelling, membrane potential dissipation and organelle release of the pro-apoptotic protein cytochrome c. JM-20 also inhibited the mitochondrial hydrolytic activity of F1F0-ATP synthase and Ca2+ influx. Therefore, JM-20 may be a multi-target neuroprotective agent, promoting reductions in neuronal excitotoxic injury and the protection of the mitochondria from Ca2+-induced impairment as well as the preservation of cellular energy balance.

The effects of JM-20 on the glutamatergic system in synaptic vesicles, synaptosomes and neural cells cultured from rat brain

“…We observed here that JM-20 enhanced glutamate uptake in astrocyte culture alone at very low micromolar concentrations, but it did not, in cortical neurons. Because the astrocytic glutamate transport plays a major role in maintaining extracellular glutamate concentrations below neurotoxic levels (Anderson and Swanson, 2000;Danbolt, 2001), and the neurotransmitter uptake by astrocytes can be stimulated by several neuroprotective compounds (Beller et al, 2011;Karki et al, 2014;Wu et al, 2008), the above mentioned effects of JM-20 may contribute to explain its anti-excitotoxic actions in vitro (Nuñez-Figueredo et al, 2014a) and in vivo (Nuñez-Figueredo et al, 2014b).…”

“…In our previous study, we showed that JM-20 inhibits the hydrolytic activity of F1FO-ATP synthase in mitochondria and submitochondrial particles isolated from rat liver (Nuñez-Figueredo et al, 2014a). Considering that F-ATPases and V-ATPases have structural and functional similarities, we hypothesized that this molecule may inhibit V-ATPase activity.…”

“…The glutamate levels in JM-20-treated rats were equivalent to normal values because no difference was observed when compared to those from a sham-operated group (4.5 ± 1.2 μM). Additionally, we identified the ability of this molecule to selectively inhibit the hydrolytic activity of mitochondrial F1FO ATP synthase at very low micromolar concentrations (IC50 = 5 μM) (Nuñez-Figueredo et al, 2014a). Because V-ATPase and mitochondrial F-type ATPase (F-ATPase) have a similar structure and mechanism of action (Nelson and Harvey, 1999), we hypothesized that the in vivo anti-excitotoxic effect of JM-20 observed elsewhere could be mediated by the inhibition of V-ATPase activity and the subsequent reduction in quantum release of glutamate into the synaptic cleft.…”

“…Radioactivity was measured with a Wallac Model 1409 liquid scintillation counter (PerkinElmer-Wallac, Inc., Gaithersburg, Md). JM-20 was added at final concentrations of 0.1, 1, and 10 μM, based on previous results (Nuñez-Figueredo et al, 2014a).…”

Antioxidant and Neuroprotective Effects of KM-34, A Novel Synthetic Catechol, Against Oxidative Stress-Induced Neurotoxicity

“…After MTT withdrawal, 200 µl of DMSO was added to each well to solubilize the formazan crystals, and the absorbance of each well was measured at 540 nm using a microplate reader (POLARstar Omega fluorescence spectrophotometer, Germany). The results are expressed as the percentage of absorbance relative to the undamaged control cells [15].…”