Design and synthesis of novel 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridines as small molecule BACE-1 inhibitors

Alzheimer disease (AD) is a neuronal dementia for which no treatment has been consolidated yet. Major pathologic hallmark of AD is the aggregated extracellular amyloid-β plaques in the brains of disease sufferers. Aβ-peptide is a major component of amyloid plaques and is produced from amyloid precursor protein (APP) via the proteolysis action. An aspartyl protease known as β-site amyloid precursor protein cleaving enzyme (BACE-1) is responsible for this proteolytic action. Distinctive role of BACE-1 in AD pathogenesis has made it a validated target to develop anti-Alzheimer agents. Our structure-based virtual screening method led to the synthesis of novel 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridine BACE-1 inhibitors (6a–6p; in vitro hits). Molecular docking and DFT-based ab initio studies using B3LYP functional in association with triple-ζ basis set (TZV) proposed binding mode and binding energies of ligands in the active site of the receptor. In vitro BACE-1 inhibitory activities were determined by enzymatic fluorescence resonance energy transfer (FRET) assay. Most of the synthesized dihydropyridine scaffolds were active against BACE-1 while 6d, 6k, 6n and 6a were found to be the most potent molecules with IC₅₀ values of 4.21, 4.27, 4.66 and 6.78 μM, respectively. Superior BACE-1 inhibitory activities were observed for dihydropyridine derivatives containing fused/nonfused thiazole containing groups, possibly attributing to the additional interactions with S2–S3 subpocket residues. Relatively reliable correlation between calculated binding energies and experimental BACE-1 inhibitory activities was achieved (R² = 0.51). Moreover, compounds 6d, 6k, 6n and 6a exhibited relatively no calcium channel blocking activity with regard to nifedipine suggesting them as appropriate candidates for further modification(s) to BACE-1 inhibitory scaffolds.     

RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos

Heterotrimeric G proteins are crucial for asymmetric cell division, but the mechanisms of signal activation remain poorly understood. Here, we establish that the evolutionarily conserved protein RIC-8 is required for proper asymmetric division of one-cell stage C. elegans embryos. Spindle severing experiments demonstrate that RIC-8 is required for generation of substantial pulling forces on astral microtubules. RIC-8 physically interacts with GOA-1 and GPA-16, two Galpha subunits that act in a partially redundant manner in one-cell stage embryos. RIC-8 preferentially binds to GDP bound GOA-1 and is a guanine nucleotide exchange factor (GEF) for GOA-1. Our analysis suggests that RIC-8 acts before the GoLoco protein GPR-1/2 in the sequence of events leading to Galpha activation. Furthermore, coimmunoprecipitation and in vivo epistasis demonstrate that inactivation of the Gbeta subunit GPB-1 alleviates the need for RIC-8 in one-cell stage embryos. Our findings suggest a mechanism in which RIC-8 favors generation of Galpha free from Gbetagamma and enables GPR-1/2 to mediate asymmetric cell division.     

Polarity mediates asymmetric trafficking of the Gbeta heterotrimeric G-protein subunit GPB-1 in C. elegans embryos

Asymmetric cell division is an evolutionarily conserved process that gives rise to daughter cells with different fates. In one-cell stage C. elegans embryos, this process is accompanied by asymmetric spindle positioning, which is regulated by anterior-posterior (A-P) polarity cues and driven by force generators located at the cell membrane. These force generators comprise two Gα proteins, the coiled-coil protein LIN-5 and the GoLoco protein GPR-1/2. The distribution of GPR-1/2 at the cell membrane is asymmetric during mitosis, with more protein present on the posterior side, an asymmetry that is thought to be crucial for asymmetric spindle positioning. The mechanisms by which the distribution of components such as GPR-1/2 is regulated in time and space are incompletely understood. Here, we report that the distribution of the Gβ subunit GPB-1, a negative regulator of force generators, varies across the cell cycle, with levels at the cell membrane being lowest during mitosis. Furthermore, we uncover that GPB-1 trafficks through the endosomal network in a dynamin- and RAB-5-dependent manner, which is most apparent during mitosis. We find that GPB-1 trafficking is more pronounced on the anterior side and that this asymmetry is regulated by A-P polarity cues. In addition, we demonstrate that GPB-1 depletion results in the loss of GPR-1/2 asymmetry during mitosis. Overall, our results lead us to propose that modulation of Gβ trafficking plays a crucial role during the asymmetric division of one-cell stage C. elegans embryos.     

Using a competent tissue for efficient transformation of sugarbeet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

Summary Despite intensive efforts, a reproducible and reliable method for transformation of sugarbeet plants is still lacking. Having examined several explants, we found that cells around the main vein of leaves of plantlets reared from tissue-cultured apical meristems are sufficiently competent for transformation and subsequent regeneration. A transformation protocol was designed by evaluating alterations in several parameters such as plant genotype, Agrobacterium strain, antibiotics, darkness and duration of co-culture period. An average transformation rate of 6.2% transformed shoots per explant was achieved as judged by Southern blotting. Consistent inactivation of reporter genes was correlated to multiple copies of transgenes present in some transformants. The necessary steps for rooting and planting of transformed shoots were also established.     

Cortical localization of the Galpha protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the Galpha proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using co-immunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the Gbeta protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two Galpha proteins can activate the same pathway and that their dual presence is normally needed to counter Gbetagamma. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA-16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between Galpha proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.     

Genetic diversity measures of local European beef cattle breeds for conservation purposes

This study was undertaken to determine the genetic structure, evolutionary relationships, and the genetic diversity among 18 local cattle breeds from Spain, Portugal, and France using 16 microsatellites. Heterozygosities, estimates of Fst, genetic distances, multivariate and diversity analyses, and assignment tests were performed. Heterozygosities ranged from 0.54 in the Pirenaica breed to 0.72 in the Barrosã breed. Seven percent of the total genetic variability can be attributed to differences among breeds (mean F_st = 0.07; P < 0.01). Five different genetic distances were computed and compared with no correlation found to be significantly different from 0 between distances based on the effective size of the population and those which use the size of the alleles. The Weitzman recursive approach and a multivariate analysis were used to measure the contribution of the breeds diversity. The Weitzman approach suggests that the most important breeds to be preserved are those grouped into two clusters: the cluster formed by the Mirandesa and Alistana breeds and that of the Sayaguesa and Tudanca breeds. The hypothetical extinction of one of those clusters represents a 17% loss of diversity. A correspondence analysis not only distinguished four breed groups but also confirmed results of previous studies classifying the important breeds contributing to diversity. In addition, the variation between breeds was sufficiently high so as to allow individuals to be assigned to their breed of origin with a probability of 99% for simulated samples.     

Polymorphisms in the <Emphasis Type="Italic">HSP90AA1</Emphasis> 5′ flanking region are associated with scrapie incubation period in sheep

Susceptibility to scrapie is mainly controlled by point mutations at the PRNP locus. However, additional quantitative trait loci (QTL) have been identified across the genome including a region in OAR18. The gene which encodes the inducible form of the cytoplasmic Hsp90 chaperone (HSP90AA1) maps within this region and seems to be associated with the resistance/susceptibility to scrapie in sheep. Here, we have analyzed several polymorphisms which were previously described in the ovine HSP90AA1 5′ flanking region and in intron 10 in two naturally scrapie infected Romanov sheep populations. First, we have studied 58 ARQ/VRQ animals pertaining to the sire family where the QTL influencing scrapie incubation period in OAR18 was detected. We have found a significant association between polymorphisms localized at −660 and −528 in the HSP90AA1 5′ flanking region and the scrapie incubation period. These two polymorphisms have also been studied in a second sample constituted by 62 VRQ/VRQ sheep showing an extreme incubation period. Results are concordant with the first dataset. Finally, we have studied the HSP90AA1 expression in scrapie and control animals (N = 41) with different HSP90AA1 genotypes by real time PCR on blood samples. The HSP90AA1 expression rate was equivalent in CC₋₆₀₀AA₋₅₂₈ and CG₋₆₀₀AG₋₅₂₈ scrapie resistant animals (ARR/ARR) and was higher in their CC₋₆₀₀AA₋₅₂₈ than in their CG₋₆₀₀AG₋₅₂₈ scrapie susceptible counterparts (VRQ/VRQ). Our results support the hypothesis that the ovine HSP90AA1 gene acts as a modulator of scrapie susceptibility, contributing to the observed differences in the incubation period of scrapie infected animals with the same PRNP genotype.     

A new pharmacological role for donepezil: attenuation of morphine-induced tolerance and apoptosis in rat central nervous system

Background

Tolerance to the analgesic effect of opioids is a pharmacological phenomenon that occurs after their prolonged administration. It has been shown that morphine-induced tolerance is associated with apoptosis in the central nervous system and neuroprotective agents which prevented apoptosis signaling could attenuate tolerance to the analgesic effects. On the other hand donepezil, an acetylcholinesterase inhibitor, has been reported to have neuroprotective effects. Therefore in this study, the effect of systemic administration of donepezil on morphine-induced tolerance and apoptosis in the rat cerebral cortex and lumbar spinal cord was evaluated. Various groups of rats received morphine (ip) and different doses of donepezil (0, 0.5, 1, 1.5 mg/kg/day). Nociception was assessed using tail flick apparatus. Tail flick latency was recorded when the rat shook its tail. For apoptosis assay other groups of rats received the above treatment and apoptosis was evaluated by in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method.

Results

The results showed that administration of donepezil (0.5, 1, 1.5 mg/kg, ip) delayed the morphine tolerance for 9, 12 and 17 days, respectively. Furthermore pretreatment injection of donepezil attenuated the number of apoptotic cells in the cerebral cortex and lumbar spinal cord compared to the control group.

Conclusion

In conclusion, we found that systemic administration of donepezil attenuated morphine-induced tolerance and apoptosis in the rat cerebral cortex and lumbar spinal cord.