Generation, optimization and characterization of novel anti-prion compounds.

Prions are misfolded proteins involved in neurodegenerative diseases of high interest in veterinary and public health. In this work, we report the chemical space exploration around the anti-prion compound BB 0300674 in order to gain an understanding of its Structure Activity Relationships (SARs). A series of 43 novel analogues, based on four different chemical clusters, were synthetized and tested against PrPSc and mutant PrP toxicity assays. From this biological screening, two compounds (59 and 65) emerged with a 10-fold improvement in anti-prion activity compared with the initial lead compound, presenting at the same time interesting cell viability.

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using In Silico Screening Delay the Progression of an Illness in Prion-Infected Mice.

The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the "hot spot," stabilizes the structure of PrPC and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrPSc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.

Post-ischemic treatment with toki-shakuyaku-san (tang-gui-shao-yao-san) prevents the impairment of spatial memory induced by repeated cerebral ischemia in rats.

Previously we have reported that Toki-shakuyaku-san (TSS) ameliorated the impairment of spatial memory induced by single cerebral ischemia (1 x 10 minutes) and scopolamine, a muscarinic receptor antagonist. In this experiment, we studied the effect of TSS on repeated cerebral ischemia (2 x 10 minutes, 1-hour interval) induced impairment of spatial memory and neuronal injury in rats. The 8-day post-ischemic treatment with TSS (30-300 mg/kg) was administered p.o. once per day. TSS dose-dependently prevented the impairment of spatial memory, neuronal death and TUNEL positive cells induced by repeated cerebral ischemia. In order to determine the mechanism of TSS, we also studied the effect of TSS on GluR2 mRNA, one of the glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor subunits. Repeated cerebral ischemia significantly decreased GluR2 flop mRNA at 1 and 3 days after the occlusion. TSS (300 mg/kg) significantly suppressed the decrease in GluR2 flop at 3 days after repeated cerebral ischemia. These results suggested that the TSS has neuroprotective action which may be indirectly mediated by the AMPA receptor, and TSS may be beneficial for the treatment of cerebrovascular dementia.

Protective effect of buckwheat polyphenols against long-lasting impairment of spatial memory associated with hippocampal neuronal damage in rats subjected to repeated cerebral ischemia.

In the present experiment, we studied the action of buckwheat polyphenol (BWP, from Fagopyrum esculentum MOENCH) in a repeated cerebral ischemia model, which induced a strong and long-lasting impairment of spatial memory in 8-arm radial maze with hippocampal CA1 cell death in rats. BWP (600 mg/kg, continuous 21-day p.o.) significantly ameliorated not only the impairment of spatial memory in the 8-arm radial maze, but also necrosis and TUNEL-positive cells in the hippocampal CA1 area subjected to repeated cerebral ischemia (10 min x 2 times occlusion, 1-h interval) in rats. In order to investigate the mechanism of BWP protective action, we measured the release of glutamate and NO(x)(-) (NO(2)(-) + NO(3)(-)) production induced by repeated cerebral ischemia in the rat dorsal hippocampus using microdialysis. A 14-day BWP treatment significantly inhibited the excess release of glutamate after the second occlusion. In addition, the BWP remarkably suppressed a delayed increase in NO(x)(-) (NO(2)(-) + NO(3)(-)) induced by repeated cerebral ischemia in the dorsal hippocampus as determined in vivo by microdialysis. However, the 14-day treatment did not affect hippocampal blood flow in either intact rats or rats subjected to repeated ischemia measured by lasser Doppler flowmeter. These results suggested that BWP might ameliorate spatial memory impairment by inhibiting glutamate release and the delayed generation of NO(x)(-) in rats subjected to repeated cerebral ischemia.