N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia.
The Rh-catalyzed hydroacylative union of aldehydes and o-alkynyl anilines leads to 2-aminophenyl enones, and onward to substituted quinolines. The mild reaction conditions employed in this chemistry result in a process that displays broad functional group tolerance, allowing the preparation of diversely substituted quinolines in high yields. Extension to the use of o-alkynyl nitro arenes as substrates leads to 2-nitrochalcones, from which both quinolines and quinoline N-oxides can be accessed.
Biochemical
assay interference is becoming increasingly recognized
as a significant waste of resource in drug discovery, both in industry
and academia. A seminal publication from Baell and Holloway raised
the awareness of this issue, and they published a set of alerts to
identify what they described as PAINS (pan-assay interference compounds).
These alerts have been taken up by drug discovery groups, even though
the original paper had a somewhat limited data set. Here, we have
taken Lilly’s far larger internal data set to assess the PAINS
alerts on four criteria: promiscuity (over six assay formats including
AlphaScreen), compound stability, cytotoxicity, and presence of a
high Hill slope as a surrogate for non-1:1 protein–ligand binding.
It was found that only three of the alerts show pan-assay promiscuity,
and the alerts appear to encode primarily AlphaScreen promiscuous
molecules. Although not enriching for pan-assay promiscuity, many
of the alerts do encode molecules that are unstable, show cytotoxicity,
and increase the prevalence of high Hill slopes.
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