Using fragment-based screening of a focused fragment library, 2-aminoquinoline 1 was identified as an initial hit for BACE1. Further SAR development was supported by X-ray structures of BACE1 cocrystallized with various ligands and molecular modeling studies to expedite the discovery of potent compounds. These strategies enabled us to integrate the C-3 side chain on 2-aminoquinoline 1 extending deep into the P2′ binding pocket of BACE1 and enhancing the ligand's potency. We were able to improve the BACE1 potency to subnanomolar range, over 106-fold more potent than the initial hit (900 μM). Further elaboration of the physical properties of the lead compounds to those more consistent with good blood–brain barrier permeability led to inhibitors with greatly improved cellular activity and permeability. Compound 59 showed an IC50 value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced into rat pharmacokinetic and pharmacodynamic studies and demonstrated significant reduction of Aβ levels in cerebrospinal fluid (CSF).
Recent studies implicate dendritic endocannabinoid release from subsynaptic dendrites and subsequent inhibition of neurotransmitter release from nerve terminals as a means of retrograde signaling in multiple brain regions. Here we show that type 1 cannabinoid receptor-mediated endocannabinoid signaling is not involved in the retrograde control of synaptic efficacy at inhibitory synapses between fast-spiking interneurons and pyramidal cells in layer 2/3 of the neocortex. Vesicular neurotransmitter transporters, such as vesicular glutamate transporters (VGLUTs) 1 and 2, are localized to presynaptic terminals and accumulate neurotransmitters into synaptic vesicles. A third subtype of VGLUTs (VGLUT3) was recently identified and found localized to dendrites of various cell types. We demonstrate, using multiple immunofluorescence labeling and confocal laser-scanning microscopy, that VGLUT3-like immunoreactivity is present in dendrites of layer 2/3 pyramidal neurons in the rat neocortex. Electron microscopy analysis confirmed that VGLUT3-like labeling is localized to vesicular structures, which show a tendency to accumulate in close proximity to postsynaptic specializations in dendritic shafts of pyramidal cells. Dual whole-cell recordings revealed that retrograde signaling between fast-spiking interneurons and pyramidal cells was enhanced under conditions of maximal efficacy of VGLUT3-mediated glutamate uptake, whereas it was reduced when glutamate uptake was inhibited by incrementing concentrations of the nonselective VGLUT inhibitor Evans blue (0.5-5.0 μ m ) or intracellular Cl - concentrations (4-145 m m ). Our results present further evidence that dendritic vesicular glutamate release, controlled by novel VGLUT isoforms, provides fast negative feedback at inhibitory neocortical synapses, and demonstrate that glutamate can act as a retrograde messenger in the CNS.
Abstract: The role of dopaminergic innervation on the postnatal developmental expression of D 1 dopamine receptors was investigated. Bilateral destruction of dopa‐mine‐containing neurons was achieved by treating rats intracisternally with 6‐hydroxydopamine (6‐OHDA) on postnatal day 3, and rats were killed on day 21. To ensure effective reduction of D 1 receptor activation by residual dopamine, a group of 6‐OHDA‐lesioned rats was given twice daily injections of the D 1 receptor antagonist SCH‐23390, from day 4 to 20. D 1 dopamine receptor binding was assessed in the caudate—putamen, nucleus accumbens, and olfactory tubercle by quantitative autoradiographic analysis of [ 3 H]SCH‐23390 binding. In addition, the relative amount of D 1A receptor mRNA was assessed by in situ hybridization of a 35 S‐labeled riboprobe. In the developing rats, neither the amount of [ 3 H]SCH‐23390 binding nor the amount of D 1A receptor mRNA was altered by 6‐OHDA lesioning followed by chronic treatment with SCH‐23390. Thus, bilateral destruction of dopamine‐containing neurons and treatment with SCH‐23390 in neonatal rats did not interfere with the developmental expression of D 1 receptors or alter the levels of mRNA that code for this receptor protein. Treatment of intact rats with SCH‐23390 from postnatal day 4 to 20 also did not alter [ 3 H]SCH‐23390 binding or levels of D 1 receptor mRNA. However, adult rats treated chronically with SCH‐23390 exhibited increased [ 3 H]SCH‐23390 binding but did not show a significant change in D 1 receptor mRNA levels.
