Prenylated Quinolinecarboxylic Acid Derivative Prevents Neuronal Cell Death Through Inhibition of MKK4

The development of neuroprotective agents is necessary for the treatment of neurodegenerative diseases. Here, we report PQA-11, a prenylated quinolinecarboxylic acid (PQA) derivative, as a potent neuroprotectant. PQA-11 inhibits glutamate-induced cell death and caspase-3 activation in hippocampal cultures, as well as inhibits N-Methyl-4-phenylpyridinium iodide- and amyloid β1-42-induced cell death in SH-SY5Y cells. PQA-11 also suppresses mitogen-activated protein kinase kinase 4 (MKK4) and c-jun N-terminal kinase (JNK) signaling activated by these neurotoxins. Quartz crystal microbalance analysis and in vitro kinase assay reveal that PQA-11 interacts with MKK4, and inhibits its sphingosine-induced activation. The administration of PQA-11 by intraperitoneal injection alleviates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of nigrostriatal dopaminergic neurons in mice. These results suggest that PQA-11 is a unique MKK4 inhibitor with potent neuroprotective effects in vitro and in vivo. PQA-11 may be a valuable lead for the development of novel neuroprotectants

Oxygen consumption induced by Ppc-1.

<p>(A) Enhanced oxygen consumption was induced by Ppc-1 in a dose-dependent manner. Mitochondria-enriched fraction (0.1 mg protein) in assay buffer was pre-incubated at 30°C for 0.5 min, and the indicated amounts of Ppc-1 were added to the mixture at time point 0 min. Recording of oxygen level was started at 0.5 min after the addition of the compound, and then an aliquot of ADP was added to a final concentration of 400 μM to induce ‘state 3’. (B) Effects of oligomycin A (0.75 μM) and KCN (1 mM) on the oxygen consumption were examined. (C, D) The oxygen consumption rate in the absence (C) or presence (D) of ADP was calculated. Ppc-1 showed a significant increase in oxygen consumption at concentrations above 10 μM, but no effect on ‘state 3’ at any dose tested. **<i>p</i> < 0.01. (E) The RCR for ADP was calculated, confirming that Ppc-1 acts as a mitochondrial uncoupler. (F) Effect of Ppc-1 on the oxygen consumption rate was examined in assay buffer containing various concentrations of ADP. A representative reciprocal plot is shown, and the <i>K</i>m values for ADP are inserted.</p

Screening of uncoupling agents.

<p>(A) Structures of small molecule compounds identified in slime molds. (B) Effects of compounds (20 μM each) on oxygen consumption were measured in the absence or presence (state 3) of ADP using isolated mitochondria. Four compounds were found to enhance oxygen consumption, suggesting uncoupling activity. Only Ppc-1 exhibited no inhibitory effect on ‘state 3’.</p

Profiling of Serum Factors.

<p>*: <i>p</i><0.05.</p><p>Profiling of Serum Factors.</p

Weight Loss by Ppc-1, a Novel Small Molecule Mitochondrial Uncoupler Derived from Slime Mold

<div><p>Mitochondria play a key role in diverse processes including ATP synthesis and apoptosis. Mitochondrial function can be studied using inhibitors of respiration, and new agents are valuable for discovering novel mechanisms involved in mitochondrial regulation. Here, we screened small molecules derived from slime molds and other microorganisms for their effects on mitochondrial oxygen consumption. We identified Ppc-1 as a novel molecule which stimulates oxygen consumption without adverse effects on ATP production. The kinetic behavior of Ppc-1 suggests its function as a mitochondrial uncoupler. Serial administration of Ppc-1 into mice suppressed weight gain with no abnormal effects on liver or kidney tissues, and no evidence of tumor formation. Serum fatty acid levels were significantly elevated in mice treated with Ppc-1, while body fat content remained low. After a single administration, Ppc-1 distributes into various tissues of individual animals at low levels. Ppc-1 stimulates adipocytes in culture to release fatty acids, which might explain the elevated serum fatty acids in Ppc-1-treated mice. The results suggest that Ppc-1 is a unique mitochondrial regulator which will be a valuable tool for mitochondrial research as well as the development of new drugs to treat obesity.</p></div

Release of fatty acids from 3T3-L1 cells by Ppc-1. Lipid extracts in the culture supernatants of differentiated 3T3-L1 cells treated with 10 μM Ppc-1 for 48 h or 72 h were analyzed by HPLC-MS/MS system.

<p>A. A representative analysis of total fatty acids (A), and fatty acid components (B) (72 h only) is shown. The peak areas were calculated, and results are expressed as mean and SD. **<i>p</i> < 0.01.</p

Examination of Ppc-1-treated mice.

<p>(A) Cross sections were prepared of fixed kidney and liver tissues from mice in the control and Ppc-1 treated (0.8 mg/week/kg) groups and stained with hematoxylin-eosin. Representative images are shown; no significant differences were detected between the two groups. (B) Daily food intake in 4 groups was recorded throughout the experiments. (C) Body surface temperature was monitored with an infrared thermometer following a single injection of Ppc-1 (0.8 mg/kg).</p