The 3C-like proteinase (3CL(pro)) of severe acute respiratory syndrome (SARS) coronavirus is a key target for structure-based drug design against this viral infection. The enzyme recognizes peptide substrates with a glutamine residue at the P1 site. A series of keto-glutamine analogues with a phthalhydrazido group at the alpha-position were synthesized and tested as reversible inhibitiors against SARS 3CL(pro). Attachment of tripeptide (Ac-Val-Thr-Leu) to these glutamine-based "warheads" generated significantly better inhibitors (4a-c, 8a-d) with IC(50) values ranging from 0.60 to 70 microM.

Abstract. Shafaati M, Olin M, Bå vner A, Pettersson H, Rozell B, Meaney S, Parini P, Björkhem I (Karolinska University Hospital Huddinge, Huddinge, Sweden; Dublin Institute of Technology, Dublin, Ireland). Enhanced production of 24S-hydroxycholesterol is not sufficient to drive liver X receptor target genes in vivo. J Intern Med 2011; 270: 377-387.Background. Oxysterols such as 24S-hydroxycholesterol (OHC) and 27-OHC are intermediates of cholesterol excretion pathways. In addition, they are putative endogenous agonists of the liver X receptor (LXR) class of nuclear hormone receptors and are thought to be important mediators of cholesterol-dependent gene regulation. 24S-OHC is one of the most efficient endogenous LXR agonists known and is present in the brain and in the circulation at relatively high levels.

The 3C-like protease (3CLpro), which controls the severe acute respiratory syndrome (SARS) coronavirus replication, has been identified as a potential target for drug design in the treatment of SARS. A series of tetrapeptide phthalhydrazide ketones, pyridinyl esters, and their analogs have been designed, synthesized, and evaluated as potential SARS 3CLpro inhibitors. Some pyridinyl esters are identified as very potent inhibitors, with IC50 values in the nanomolar range (50-65 nM). Electrospray mass spectrometry indicates a mechanism involving acylation of the active site cysteine thiol for this class of inhibitors.

No abstract

The large pool of cholesterol in the brain is effi ciently isolated from other pools of cholesterol in the body by the effi cient blood-brain barrier (for reviews, see 1, 2 ). In contrast to the lipoprotein-bound cholesterol in the circulation, side-chain oxidized cholesterol metabolites are able to cross the blood-brain barrier and fl uxes of oxysterols have been demonstrated in both directions. We ( 3, 4 ) and others ( 5 ) have shown that about two-thirds of the synthesis of cholesterol in the brain is compensated for by a fl ow of 24S-hydroxycholesterol (24OHC) from the brain into the circulation. We also demonstrated a fl ux of the extracerebral metabolite 27-hydroxycholesterol (27OHC) from the circulation into the brain with a net uptake of this oxysterol ( 6 ). Most of the 27OHC present in the cerebrospinal fl uid originates from the circulation ( 7 ).Despite the substantial uptake of 27OHC by the brain, the levels of this oxysterol in the brain are very low, reaching only 5-10% of the levels of 24OHC ( 3,8 ). The reason for this is a highly effi cient metabolism. We have shown that the end metabolite of 27OHC in the brain is a steroid acid, 7 ␣ -hydroxy-3-oxo-4-cholestenoic acid, and that there is a continuous fl ux of this acid from the brain into the circulation ( 9 ). The nature of this acid as an end metabolite is illustrated by a very substantial accumulation of it in encapsulated brain hematomas ( 10 ). The rate-limiting step in the conversion of 27OHC into the steroid acid appears to be the introduction of a 7 ␣ -hydroxyl group catalyzed by the enzyme oxysterol 7 ␣ -hydroxylase, CYP7B1 ( 9 ). This enzyme is exclusively located in neuronal cells in the brain ( 11 ).Despite the fact that lipoprotein-bound cholesterol does not pass the blood-brain barrier ( 1, 2 ), hypercholesAbstract There is a signifi cant fl ux of the neurotoxic oxysterol 27-hydroxycholesterol (27OHC) from the circulation across the blood-brain barrier. Because there is a correlation between 27OHC and cholesterol in the circulation and lipoprotein-bound cholesterol does not pass the blood-brain barrier, we have suggested that 27OHC may mediate the effects of hypercholesterolemia on the brain. We previously demonstrated a modest accumulation of 27OHC in brains of patients with sporadic Alzheimer's disease (AD), consistent with a role of 27OHC as a primary pathogenetic factor. We show here that there is a 4-fold accumulation of 27OHC in different regions of the cortexes of patients carrying the Swedish amyloid precursor protein (APPswe) 670/671 mutation. The brain levels of sitosterol and campesterol were not signifi cantly different in the AD patients compared with the controls, suggesting that the blood-brain barrier was intact in the AD patients. We conclude that accumulation of 27OHC is likely to be secondary to neurodegeneration, possibly a result of reduced activity of CYP7B1, the neuronal enzyme responsible for metabolism of 27OHC. We discuss the possibility of a vicious circle in the brains of the patients with familial AD wh...

No abstract

Expression of the Vglut2/Slc17a6 gene encoding the Vesicular glutamate transporter 2 (VGLUT2) in midbrain dopamine (DA) neurons enables these neurons to co-release glutamate in the nucleus accumbens (NAc), a feature of putative importance to drug addiction. For example, it has been shown that conditional deletion of Vglut2 gene expression within developing DA neurons in mice causes altered locomotor sensitization to addictive drugs, such as amphetamine and cocaine, in adulthood. Alterations in DA neurotransmission in the mesoaccumbal pathway has been proposed to contribute to these behavioral alterations but the underlying molecular mechanism remains largely elusive. Repeated exposure to cocaine is known to cause lasting adaptations of excitatory synaptic transmission onto medium spiny neurons (MSNs) in the NAc, but the putative contribution of VGLUT2-mediated glutamate co-release from the mesoaccumbal projection has never been investigated. In this study, we implemented a tamoxifen-inducible Cre-LoxP strategy to selectively probe VGLUT2 in mature DA neurons of adult mice. Optogenetics-coupled patch clamp analysis in the NAc demonstrated a significant reduction of glutamatergic neurotransmission, whilst behavioral analysis revealed a normal locomotor sensitization to amphetamine and cocaine. When investigating if the reduced level of glutamate co-release from DA neurons caused a detectable post-synaptic effect on MSNs, patch clamp analysis identified an enhanced baseline AMPA/NMDA ratio in DA receptor subtype 1 (DRD1)-expressing accumbal MSNs which occluded the effect of cocaine on synaptic transmission. We conclude that VGLUT2 in mature DA neurons actively contributes to glutamatergic neurotransmission in the NAc, a finding which for the first time highlights VGLUT2-mediated glutamate co-release in the complex mechanisms of synaptic plasticity in drug addiction.

Transgenic mouse lines are instrumental in our attempt to understand brain function. Promoters driving transgenic expression of the gene encoding Cre recombinase are crucial to ensure selectivity in Cre-mediated targeting of floxed alleles using the Cre-Lox system. For the study of dopamine (DA) neurons, promoter sequences driving expression of the Dopamine transporter (Dat) gene are often implemented and several DAT-Cre transgenic mouse lines have been found to faithfully direct Cre activity to DA neurons. While evaluating an established DAT-Cre mouse line, reporter gene expression was unexpectedly identified in cell somas within the amygdala. To indiscriminately explore Cre activity in DAT-Cre transgenic lines, systematic whole-brain analysis of two DAT-Cre mouse lines was performed upon recombination with different types of floxed reporter alleles. Results were compared with data available from the Allen Institute for Brain Science. The results identified restricted DAT-Cre-driven reporter gene expression in cell clusters within several limbic areas, including amygdaloid and mammillary subnuclei, septum and habenula, areas classically associated with glutamatergic and GABAergic neurotransmission. While no Dat gene expression was detected, ample co-localization between DAT-Cre-driven reporter and markers for glutamatergic and GABAergic neurons was found. Upon viral injection of a fluorescent reporter into the amygdala and habenula, distinct projections from non-dopaminergic DAT-Cre neurons could be distinguished. The study demonstrates that DAT-Cre transgenic mice, beyond their usefulness in recombination of floxed alleles in DA neurons, could be implemented as tools to achieve selective targeting in restricted excitatory and inhibitory neuronal populations within the limbic neurocircuitry.