11 research outputs found
Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474
A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomicmethods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system
Effects of Shock and Vibration on Product Quality during Last-Mile Transportation of Ebola Vaccine under Refrigerated Conditions
Analyzing vaccine stability under different storage and transportation conditions is critical to ensure that effectiveness and safety are not affected by distribution. In a simulation of the last mile in the supply chain, we found that shock and vibration had no effect on Ad26.ZEBOV/MVA-BN-Filo Ebola vaccine regimen quality under refrigerated conditions
Cellular Assay to Study β-Arrestin Recruitment by the Cannabinoid Receptors 1 and 2
Cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G protein pathway and the recruitment of β-arrestins. Differences in the activation level of these pathways lead to biased signaling. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1R and CB2R using the PathHunter® assay. This is a cellular assay that uses a β-galactosidase complementation system which has a chemiluminescent read-out and can be performed in 384-well plates. We have successfully used this assay to characterize a set of reference ligands (both agonists, antagonists, and an inverse agonist) on human CB1R and CB2R, of which some examples will be presented here
Removal of Human Ether-à-go-go Related Gene (hERG) K<sup>+</sup> Channel Affinity through Rigidity: A Case of Clofilium Analogues
Cardiotoxicity
is a side effect that plagues modern drug design and is very often
due to the off-target blockade of the human ether-à-go-go related
gene (hERG) potassium channel. To better understand the structural
determinants of this blockade, we designed and synthesized a series
of 40 derivatives of clofilium, a class III antiarrhythmic agent.
These were evaluated in radioligand binding and patch-clamp assays
to establish structure–affinity relationships (SAR) for this
potassium channel. Efforts were especially focused on studying the
influence of the structural rigidity and the nature of the linkers
composing the clofilium scaffold. It was shown that introducing triple
bonds and oxygen atoms in the <i>n</i>-butyl linker of the
molecule greatly reduced affinity without significantly modifying
the p<i>K</i><sub>a</sub> of the essential basic nitrogen.
These findings could prove useful in the first stages of drug discovery
as a systematic way of reducing the risk of hERG K<sup>+</sup> channel
blockade-induced cardiotoxicity
Structure-kinetic relationship studies of cannabinoid CB 2 receptor agonists reveal substituent-specific lipophilic effects on residence time
R agonists can be designed to have optimal kinetic profiles, which could aid the lead optimization process in drug discovery for the study or treatment of inflammatory diseases.Molecular Physiolog
Selective Photoaffinity Probe That Enables Assessment of Cannabinoid CB2 Receptor Expression and Ligand Engagement in Human Cells
ISSN:0002-7863ISSN:1520-512
Cannabinoid CB receptor ligand profiling reveals biased signalling and off-target activity
The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research
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
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