13 research outputs found
Comprehensive Analysis of Structure–Activity Relationships of α‑Ketoheterocycles as <i>sn</i>-1-Diacylglycerol Lipase α Inhibitors
Diacylglycerol
lipase α (DAGLα) is responsible for
the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG)
in the central nervous system. DAGLα inhibitors are required
to study the physiological role of 2-AG. Previously, we identified
the α-ketoheterocycles as potent and highly selective DAGLα
inhibitors. Here, we present the first comprehensive structure–activity
relationship study of α-ketoheterocycles as DAGLα inhibitors.
Our findings indicate that the active site of DAGLα is remarkably
sensitive to the type of heterocyclic scaffold with oxazolo-4<i>N</i>-pyridines as the most active framework. We uncovered a
fundamental substituent effect in which electron-withdrawing <i>meta</i>-oxazole substituents increased inhibitor potency. (C<sub>6</sub>–C<sub>9</sub>)-acyl chains with a distal phenyl group
proved to be the most potent inhibitors. The integrated SAR data was
consistent with the proposed binding pose in a DAGLα homology
model. Altogether, our results may guide the design of future DAGLα
inhibitors as leads for molecular therapies to treat neuroinflammation,
obesity, and related metabolic disorders
Chemical Proteomics Maps Brain Region Specific Activity of Endocannabinoid Hydrolases
The
biosynthetic and catabolic enzymes of the endocannabinoids
tightly regulate endocannabinoid-mediated activation of the cannabinoid
CB<sub>1</sub> receptor. Monitoring the activities of these endocannabinoid
hydrolases in different brain regions is, therefore, key to gaining
insight into spatiotemporal control of CB<sub>1</sub> receptor-mediated
physiology. We have employed a comparative chemical proteomics approach
to quantitatively map the activity profile of endocannabinoid hydrolases
in various mouse brain regions at the same time. To this end, we used
two different activity-based probes: fluorophosphonate-biotin (FP-biotin),
which quantifies FAAH, ABHD6, and MAG-lipase activity, and MB108,
which detects DAGL-α, ABHD4, ABHD6, and ABHD12. In total, 32
serine hydrolases were evaluated in the frontal cortex, hippocampus,
striatum, and cerebellum. Comparison of endocannabinoid hydrolase
activity in the four brain regions revealed that FAAH activity was
highest in the hippocampus, and MAGL activity was most pronounced
in the frontal cortex, whereas DAGL-α was most active in the
cerebellum. Comparison of the activity profiles with a global proteomics
data set revealed pronounced differences. This could indicate that
post-translational modification of the endocannabinoid hydrolases
is important to regulate their activity. Next, the effect of genetic
deletion of the CB<sub>1</sub> receptor was studied. No difference
in the enzymatic activity was found in the cerebellum, striatum, frontal
cortex, and hippocampus of CB<sub>1</sub> receptor knockout animals
compared to wild type mice. Our results are in line with previous
reports and indicate that the CB<sub>1</sub> receptor exerts no regulatory
control over the basal production and degradation of endocannabinoids
and that genetic deletion of the CB<sub>1</sub> receptor does not
induce compensatory mechanisms in endocannabinoid hydrolase activity
Chemical Proteomics Maps Brain Region Specific Activity of Endocannabinoid Hydrolases
The
biosynthetic and catabolic enzymes of the endocannabinoids
tightly regulate endocannabinoid-mediated activation of the cannabinoid
CB<sub>1</sub> receptor. Monitoring the activities of these endocannabinoid
hydrolases in different brain regions is, therefore, key to gaining
insight into spatiotemporal control of CB<sub>1</sub> receptor-mediated
physiology. We have employed a comparative chemical proteomics approach
to quantitatively map the activity profile of endocannabinoid hydrolases
in various mouse brain regions at the same time. To this end, we used
two different activity-based probes: fluorophosphonate-biotin (FP-biotin),
which quantifies FAAH, ABHD6, and MAG-lipase activity, and MB108,
which detects DAGL-α, ABHD4, ABHD6, and ABHD12. In total, 32
serine hydrolases were evaluated in the frontal cortex, hippocampus,
striatum, and cerebellum. Comparison of endocannabinoid hydrolase
activity in the four brain regions revealed that FAAH activity was
highest in the hippocampus, and MAGL activity was most pronounced
in the frontal cortex, whereas DAGL-α was most active in the
cerebellum. Comparison of the activity profiles with a global proteomics
data set revealed pronounced differences. This could indicate that
post-translational modification of the endocannabinoid hydrolases
is important to regulate their activity. Next, the effect of genetic
deletion of the CB<sub>1</sub> receptor was studied. No difference
in the enzymatic activity was found in the cerebellum, striatum, frontal
cortex, and hippocampus of CB<sub>1</sub> receptor knockout animals
compared to wild type mice. Our results are in line with previous
reports and indicate that the CB<sub>1</sub> receptor exerts no regulatory
control over the basal production and degradation of endocannabinoids
and that genetic deletion of the CB<sub>1</sub> receptor does not
induce compensatory mechanisms in endocannabinoid hydrolase activity
Biochemical and Cellular Characterization of the Function of Fluorophosphonate-Binding Hydrolase H (FphH) in <i>Staphylococcus aureus</i> Support a Role in Bacterial Stress Response
The development of
new treatment options for bacterial
infections
requires access to new targets for antibiotics and antivirulence strategies.
Chemoproteomic approaches are powerful tools for profiling and identifying
novel druggable target candidates, but their functions often remain
uncharacterized. Previously, we used activity-based protein profiling
in the opportunistic pathogen Staphylococcus aureus to identify active serine hydrolases termed fluorophosphonate-binding
hydrolases (Fph). Here, we provide the first characterization of S. aureus FphH, a conserved, putative carboxylesterase (referred
to as yvaK in Bacillus subtilis)
at the molecular and cellular level. First, phenotypic characterization
of fphH-deficient transposon mutants revealed phenotypes
during growth under nutrient deprivation, biofilm formation, and intracellular
survival. Biochemical and structural investigations revealed that
FphH acts as an esterase and lipase based on a fold well suited to
act on a small to long hydrophobic unbranched lipid group within its
substrate and can be inhibited by active site-targeting oxadiazoles.
Prompted by a previous observation that fphH expression
was upregulated in response to fusidic acid, we found that FphH can
deacetylate this ribosome-targeting antibiotic, but the lack of FphH
function did not infer major changes in antibiotic susceptibility.
In conclusion, our results indicate a functional role of this hydrolase
in S. aureus stress responses, and hypothetical functions
connecting FphH with components of the ribosome rescue system that
are conserved in the same gene cluster across Bacillales are discussed. Our atomic characterization of FphH will facilitate
the development of specific FphH inhibitors and probes to elucidate
its physiological role and validity as a drug target
Structure-Based Design of β5c Selective Inhibitors of Human Constitutive Proteasomes
This work reports
the development of highly potent and selective
inhibitors of the β5c catalytic activity of human constitutive
proteasomes. The work describes the design principles, large hydrophobic
P3 residue and small hydrophobic P1 residue, that led to the synthesis
of a panel of peptide epoxyketones; their evaluation and the selection
of the most promising compounds for further analyses. Structure–activity
relationships detail how in a logical order the β1c/i, β2c/i,
and β5i activities became resistant to inhibition as compounds
were diversified stepwise. The most effective compounds were obtained
as a mixture of <i>cis</i>- and <i>trans</i>-biscyclohexyl
isomers, and enantioselective synthesis resolved this issue. Studies
on yeast proteasome structures complexed with some of the compounds
provide a rationale for the potency and specificity. Substitution
of the N-terminus in the most potent compound for a more soluble equivalent
led to a cell-permeable molecule that selectively and efficiently
blocks β5c in cells expressing both constitutive proteasomes
and immunoproteasomes
Image_1_Chemical Proteomic Analysis of Serine Hydrolase Activity in Niemann-Pick Type C Mouse Brain.PDF
<p>The endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the role of the ECS has not been studied yet. Most of the endocannabinoid enzymes are serine hydrolases, which can be studied using activity-based protein profiling (ABPP). Here, we report the serine hydrolase activity in brain proteomes of a NPC mouse model as measured by ABPP. Two ABPP methods are used: a gel-based method and a chemical proteomics method. The activities of the following endocannabinoid enzymes were quantified: diacylglycerol lipase (DAGL) α, α/β-hydrolase domain-containing protein 4, α/β-hydrolase domain-containing protein 6, α/β-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL α. Only the upper band corresponding to this enzyme was significantly decreased in the NPC mouse model. Chemical proteomics showed that three lysosomal serine hydrolase activities (retinoid-inducible serine carboxypeptidase, cathepsin A, and palmitoyl-protein thioesterase 1) were increased in Niemann-Pick C1 protein knockout mouse brain compared to wild-type brain, whereas no difference in endocannabinoid hydrolase activity was observed. We conclude that these targets might be interesting therapeutic targets for future validation studies.</p
Incorporation of Non-natural Amino Acids Improves Cell Permeability and Potency of Specific Inhibitors of Proteasome Trypsin-like Sites
Proteasomes degrade the majority of proteins in mammalian
cells
by a concerted action of three distinct pairs of active sites. The
chymotrypsin-like sites are targets of antimyeloma agents bortezomib
and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple
myeloma cells to these agents. Here we describe systematic effort
to develop inhibitors with improved potency and cell permeability,
yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone
(<b>4a</b>, LU-102), and a fluorescent activity-based probe
for this site. X-ray structures of <b>4a</b> and related inhibitors
complexed with yeast proteasomes revealed the structural basis for
specificity. Nontoxic to myeloma cells when used as a single agent, <b>4a</b> sensitized them to bortezomib and carfilzomib. This sensitizing
effect was much stronger than the synergistic effects of histone acetylase
inhibitors or additive effects of doxorubicin and dexamethasone, raising
the possibility that combinations of inhibitors of the trypsin-like
site with bortezomib or carfilzomib would have stronger antineoplastic
activity than combinations currently used clinically
Triazole Ureas Act as Diacylglycerol Lipase Inhibitors and Prevent Fasting-Induced Refeeding
Triazole
ureas constitute a versatile class of irreversible inhibitors
that target serine hydrolases in both cells and animal models. We
have previously reported that triazole ureas can act as selective
and CNS-active inhibitors for diacylglycerol lipases (DAGLs), enzymes
responsible for the biosynthesis of 2-arachidonoylglycerol (2-AG)
that activates cannabinoid CB<sub>1</sub> receptor. Here, we report
the enantio- and diastereoselective synthesis and structure–activity
relationship studies. We found that 2,4-substituted triazole ureas
with a biphenylmethanol group provided the most optimal scaffold.
Introduction of a chiral ether substituent on the 5-position of the
piperidine ring provided ultrapotent inhibitor <b>38</b> (DH376)
with picomolar activity. Compound <b>38</b> temporarily reduces
fasting-induced refeeding of mice, thereby emulating the effect of
cannabinoid CB<sub>1</sub>-receptor inverse agonists. This was mirrored
by <b>39</b> (DO34) but also by the negative control compound <b>40</b> (DO53) (which does not inhibit DAGL), which indicates the
triazole ureas may affect the energy balance in mice through multiple
molecular targets
Identification and Development of Biphenyl Substituted Iminosugars as Improved Dual Glucosylceramide Synthase/Neutral Glucosylceramidase Inhibitors
This
work details the evaluation of a number of N-alkylated deoxynojirimycin
derivatives on their merits as dual glucosylceramide synthase/neutral
glucosylceramidase inhibitors. Building on our previous work, we synthesized
a series of d-<i>gluco</i> and l-<i>ido</i>-configured iminosugars N-modified with a variety of
hydrophobic functional groups. We found that iminosugars featuring <i>N</i>-pentyloxymethylaryl substituents are considerably
more potent inhibitors of glucosylceramide synthase than their aliphatic
counterparts. In a next optimization round, we explored a series of
biphenyl-substituted iminosugars of both configurations (d-<i>gluco</i> and l-<i>ido</i>) with
the aim to introduce structural features known to confer metabolic
stability to drug-like molecules. From these series, two sets of molecules
emerge as lead series for further profiling. Biphenyl-substituted l-<i>ido</i>-configured deoxynojirimycin derivatives
are selective for glucosylceramidase and the nonlysosomal glucosylceramidase,
and we consider these as leads for the treatment of neuropathological
lysosomal storage disorders. Their d-<i>gluco</i>-counterparts are also potent inhibitors of intestinal glycosidases,
and because of this characteristic, we regard these as the prime candidates
for type 2 diabetes therapeutics
Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons
Diacylglycerol lipase
(DAGL)-α and -β are enzymes responsible
for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol
(2-AG). Selective and reversible inhibitors are required to study
the function of DAGLs in neuronal cells in an acute and temporal fashion,
but they are currently lacking. Here, we describe the identification
of a highly selective DAGL inhibitor using structure-guided and a
chemoproteomics strategy to characterize the selectivity of the inhibitor
in complex proteomes. Key to the success of this approach is the use
of comparative and competitive activity-based proteome profiling (ABPP),
in which broad-spectrum and tailor-made activity-based probes are
combined to report on the inhibition of a protein family in its native
environment. Competitive ABPP with broad-spectrum fluorophosphonate-based
probes and specific β-lactone-based probes led to the discovery
of α-ketoheterocycle LEI105 as a potent, highly selective, and
reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not
affect other enzymes involved in endocannabinoid metabolism including
abhydrolase domain-containing protein 6, abhydrolase domain-containing
protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase
and did not display affinity for the cannabinoid CB<sub>1</sub> receptor.
Targeted lipidomics revealed that LEI105 concentration-dependently
reduced 2-AG levels, but not anandamide levels, in Neuro2A cells.
We show that cannabinoid CB<sub>1</sub>-receptor-mediated short-term
synaptic plasticity in a mouse hippocampal slice model can be reduced
by LEI105. Thus, we have developed a highly selective DAGL inhibitor
and provide new pharmacological evidence to support the hypothesis
that “on demand biosynthesis” of 2-AG is responsible
for retrograde signaling