9 research outputs found
Chemical inhibition of fatty acid absorption and cellular uptake limits lipotoxic cell death
Chronic elevation of plasma free fatty acid (FFA) levels is commonly associated with obesity, type 2 diabetes, cardiovascular disease and some cancers. Experimental evidence indicates FFA and their metabolites contribute to disease development through lipotoxicity. Previously, we identified a specific fatty acid transport inhibitor CB16.2, a.k.a. Lipofermata, using high throughput screening methods. In this study, efficacy of transport inhibition was measured in four cell lines that are models for myocytes (mmC2C12), pancreatic Ă-cells (rnINS-1E), intestinal epithelial cells (hsCaco-2), and hepatocytes (hsHepG2), as well as primary human adipocytes. The compound was effective in inhibiting uptake with IC50s between 3 and 6 ÎŒM for all cell lines except human adipocytes (39 ÎŒM). Inhibition was specific for long and very long chain fatty acids but had no effect on medium chain fatty acids (C6-C10), which are transported by passive diffusion. Derivatives of Lipofermata were evaluated to understand structural contributions to activity. Lipofermata prevented palmitate-mediated oxidative stress, induction of BiP and CHOP, and cell death in a dose-dependent manner in hsHepG2 and rnINS-1E cells, suggesting it will prevent induction of fatty acid-mediated cell death pathways and lipotoxic disease by channeling excess fatty acids to adipose tissue and away from liver and pancreas. Importantly, mice dosed orally with Lipofermata were not able to absorb 13C-oleate demonstrating utility as an inhibitor of fatty acid absorption from the gut
Identification of Specific LigandâReceptor Interactions That Govern Binding and Cooperativity of Diverse Modulators to a Common Metabotropic Glutamate Receptor 5 Allosteric Site
A common
metabotropic glutamate receptor 5 (mGlu<sub>5</sub>) allosteric
site is known to accommodate diverse chemotypes. However, the structural
relationship between compounds from different scaffolds and mGlu<sub>5</sub> is not well understood. In an effort to better understand
the molecular determinants that govern allosteric modulator interactions
with mGlu<sub>5</sub>, we employed a combination of site-directed
mutagenesis and computational modeling. With few exceptions, six residues
(P654, Y658, T780, W784, S808, and A809) were identified as key affinity
determinants across all seven allosteric modulator scaffolds. To improve
our interpretation of how diverse allosteric modulators occupy the
common allosteric site, we sampled the wealth of mGlu<sub>5</sub> structureâactivity
relationship (SAR) data available by docking 60 ligands (actives and
inactives) representing seven chemical scaffolds into our mGlu<sub>5</sub> comparative model. To spatially and chemically compare binding
modes of ligands from diverse scaffolds, the ChargeRMSD measure was
developed. We found a common binding mode for the modulators that
placed the long axes of the ligands parallel to the transmembrane
helices 3 and 7. W784 in TM6 not only was identified as a key NAM
cooperativity determinant across multiple scaffolds, but also caused
a NAM to PAM switch for two different scaffolds. Moreover, a single
point mutation in TM5, G747V, altered the architecture of the common
allosteric site such that 4-nitro-<i>N</i>-(1,3-diphenyl-1<i>H</i>-pyrazol-5-yl)Âbenzamide (VU29) was noncompetitive with
the common allosteric site. Our findings highlight the subtleties
of allosteric modulator binding to mGlu<sub>5</sub> and demonstrate
the utility in incorporating SAR information to strengthen the interpretation
and analyses of docking and mutational data
Tetrahydronaphthyridine and Dihydronaphthyridinone Ethers As Positive Allosteric Modulators of the Metabotropic Glutamate Receptor 5 (mGlu<sub>5</sub>)
Positive
allosteric modulators (PAMs) of metabotropic glutamate
receptor 5 (mGlu<sub>5</sub>) represent a promising therapeutic strategy
for the treatment of schizophrenia. Starting from an acetylene-based
lead from high throughput screening, an evolved bicyclic dihydronaphthyridinone
was identified. We describe further refinements leading to both dihydronaphthyridinone
and tetrahydronaphthyridine mGlu<sub>5</sub> PAMs containing an alkoxy-based
linkage as an acetylene replacement. Exploration of several structural
features including western pyridine ring isomers, positional amides,
linker connectivity/position, and combinations thereof, reveal that
these bicyclic modulators generally exhibit steep SAR and within specific
subseries display a propensity for pharmacological mode switching
at mGlu<sub>5</sub> as well as antagonist activity at mGlu<sub>3</sub>. Structureâactivity relationships within a dihydronaphthyridinone
subseries uncovered <b>12c</b> (VU0405372), a selective mGlu<sub>5</sub> PAM with good in vitro potency, low glutamate fold-shift,
acceptable DMPK properties, and in vivo efficacy in an amphetamine-based
model of psychosis