Structure-Based Design and Optimization of Multitarget-Directed 2H-Chromen-2-one Derivatives as Potent Inhibitors of Monoamine Oxidase B and Cholinesterases

The multifactorial nature of Alzheimer’s disease calls for the development of multitarget agents addressing key pathogenic processes. To this end, by following a docking-assisted hybridization strategy, a number of aminocoumarins were designed, prepared, and tested as monoamine oxidases (MAOs) and acetyl- and butyryl-cholinesterase (AChE and BChE) inhibitors. Highly flexible N-benzyl-N-alkyloxy coumarins 2–12 showed good inhibitory activities at MAO-B, AChE, and BChE but low selectivity. More rigid inhibitors, bearing meta- and para-xylyl linkers, displayed good inhibitory activities and high MAO-B selectivity. Compounds 21, 24, 37, and 39, the last two featuring an improved hydrophilic/lipophilic balance, exhibited excellent activity profiles with nanomolar inhibitory potency toward hMAO-B, high hMAO-B over hMAO-A selectivity and submicromolar potency at hAChE. Cell-based assays of BBB permeation, neurotoxicity, and neuroprotection supported the potential of compound 37 as a BBB-permeant neuroprotective agent against H2O2-induced oxidative stress with poor interaction as P-gp substrate and very low cytotoxicity

Expanding Stereochemical and Skeletal Diversity Using Petasis Reactions and 1,3-Dipolar Cycloadditions

A short and modular synthetic pathway using intramolecular 1,3-dipolar cycloaddition reactions and yielding functionalized isoxazoles, isoxazolines, and isoxazolidines is described. The change in shape of previous compounds and those in this study is quantified and compared using principal moment-of-inertia shape analysis.Chemistry and Chemical Biolog

Expanding Stereochemical and Skeletal Diversity Using Petasis Reactions and 1,3-Dipolar Cycloadditions

A short and modular synthetic pathway using intramolecular 1,3-dipolar cycloaddition reactions and yielding functionalized isoxazoles, isoxazolines, and isoxazolidines is described. The change in shape of previous compounds and those in this study is quantified and compared using principal moment-of-inertia shape analysis

Modulators of Hepatic Lipoprotein Metabolism Identified in a Search for Small-Molecule Inducers of Tribbles Pseudokinase 1 Expression

Recent genome wide association studies have linked tribbles pseudokinase 1 (TRIB1) to the risk of coronary artery disease (CAD). Based on the observations that increased expression of TRIB1 reduces secretion of VLDL and is associated with lower plasma levels of LDL cholesterol and triglycerides, higher plasma levels of HDL cholesterol and reduced risk for myocardial infarction, we carried out a high throughput phenotypic screen based on quantitative RT-PCR assay to identify compounds that induce TRIB1 expression in human HepG2 hepatoma cells. In a screen of a collection of diversity-oriented synthesis (DOS)-derived compounds, we identified a series of benzofuran-based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 cDNA overexpression, as they inhibit triglyceride synthesis and apoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, key components of the lipoprotein assembly pathway. However, CRISPR-Cas9 induced chromosomal disruption of the TRIB1 locus in HepG2 cells, while confirming its regulatory role in lipoprotein metabolism, demonstrated that the effects of benzofurans persist in TRIB1-null cells indicating that TRIB1 is sufficient but not necessary to transmit the effects of the drug. Remarkably, active benzofurans, as well as natural products capable of TRIB1 upregulation, also modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR transcript and LDL receptor protein, while reducing the levels of PCSK9 transcript and secreted PCSK9 protein and stimulating LDL uptake. The effects of benzofurans are not masked by cholesterol depletion and are independent of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel class of chemically tractable small-molecule modulators that shift cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging

Structure-Based Design and Optimization of Multitarget-Directed 2<i>H</i>‑Chromen-2-one Derivatives as Potent Inhibitors of Monoamine Oxidase B and Cholinesterases

The multifactorial nature of Alzheimer’s disease calls for the development of multitarget agents addressing key pathogenic processes. To this end, by following a docking-assisted hybridization strategy, a number of aminocoumarins were designed, prepared, and tested as monoamine oxidases (MAOs) and acetyl- and butyryl-cholinesterase (AChE and BChE) inhibitors. Highly flexible <i>N</i>-benzyl-<i>N</i>-alkyloxy coumarins <b>2</b>–<b>12</b> showed good inhibitory activities at MAO-B, AChE, and BChE but low selectivity. More rigid inhibitors, bearing <i>meta</i>- and <i>para</i>-xylyl linkers, displayed good inhibitory activities and high MAO-B selectivity. Compounds <b>21</b>, <b>24</b>, <b>37</b>, and <b>39</b>, the last two featuring an improved hydrophilic/lipophilic balance, exhibited excellent activity profiles with nanomolar inhibitory potency toward hMAO-B, high hMAO-B over hMAO-A selectivity and submicromolar potency at hAChE. Cell-based assays of BBB permeation, neurotoxicity, and neuroprotection supported the potential of compound <b>37</b> as a BBB-permeant neuroprotective agent against H₂O₂-induced oxidative stress with poor interaction as P-gp substrate and very low cytotoxicity

Effects of BRD0418 on expression of selected marker genes in HepG2<i>ΔTRIB1</i>::<i>Neo</i>^<i>R</i> cell line carrying chromosomal deletion/disruption of <i>TRIB1</i> locus.

<p>(A) Schematic representation of CRISPR-Cas9 induced inactivation of <i>TRIB1</i> gene in HepG2 cells. The indicated 22 bp target sequence in exon 2 of <i>TRIB1</i> gene was used to design Cas9 guide RNA. The replacement of the target sequence in the chromosome with the Neo^R gene was induced by co-transfecting cells with the CRISPR-Cas9 plasmid together with the homologous recombination repair template consisting of Neo^R gene cassette and two ∼800 bp fragments from the <i>TRIB1</i> locus that flank the target sequence. Location of PCR primers used for identifying the Geneticin resistant clone that carries the designed chromosomal rearrangement is indicated by arrows. (B) Changes in the steady state transcript levels in HepG2<i>ΔTRIB1</i>::<i>Neo</i>^<i>R</i> cells comparing to parental HepG2 cells. The relative expression level of indicated lipid metabolic genes normalized to expression level of <i>GAPDH1</i> was measured by qRT-PCR 48 hours after plating cells in 384-well plate. The mean values for six replicates ± S.D. (<i>error bars</i>) are shown for the representative of three independent experiments. (C) Changes in transcript levels in HepG2 and in HepG2<i>ΔTRIB1</i>::<i>Neo</i>^<i>R</i> cells in response to 24 h treatment with 10 μM BRD0418. The values are normalized to DMSO controls for each strain and represent mean fold change for six replicates ± S.D. (<i>error bars</i>).</p

Marker selection of top genes differentially expressed in response to treatment with <i>TRIB1</i> expression inducers (BRD0418 and its active analogs) in comparison to treatments with <i>TRIB1</i> expression inhibitors, which included strong inhibitors (U0126 and PD-98059) and weak inhibitors (simvastatin and atorvastatin).

<p>(<i>A</i>) A heatmap of 60 genes showing strongest positive and negative difference in responses to two groups of treatments (inducers vs. inhibitors). Color scale represents logarithm fold change (LFC) values calculated relative to DMSO control treatments. The matrix analysis and visualization was carried out using GENE-E software (<a href="http://www.broadinstitute.org/cancer/software/GENE-E/index.html" target="_blank">http://www.broadinstitute.org/cancer/software/GENE-E/index.html</a>). (<i>B</i>) Chemical structures of <i>TRIB1</i> inducers from the benzofuran class profiled in the L1000 Luminex assay.</p

Effect of <i>TRIB1</i> overexpression on transcript levels of selected lipoprotein metabolic genes.

<p>Transcript levels of <i>LDLR</i>, <i>PCSK9</i>, <i>SCD1</i> and <i>APOC3</i> were measured by qRT-PCR in clones of HepG2 cells transfected with a plasmid carrying <i>TRIB1</i> gene under control of the CMV promoter. Data represent means <b>±</b> S.D. (<i>error bars</i>) for three replicates. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001 as determined with Student’s t-test for the comparison with vector control.</p