68 research outputs found
Fully Functionalized Small-Molecule Probes for Integrated Phenotypic Screening and Target Identification
Phenotypic screening offers a powerful approach to identify
small
molecules that perturb complex biological processes in cells and organisms.
The tendency of small molecules, however, to interact with multiple
protein targets, often with moderate to weak affinities, along with
the lack of straightforward technologies to characterize these interactions
in living systems, has hindered efforts to understand the mechanistic
basis for pharmacological activity. Here we address this challenge
by creating a fully functionalized small-molecule library whose membership
is endowed with: (1) one or more diversity elements to promote interactions
with different protein targets in cells, (2) a photoreactive group
for UV light-induced covalent cross-linking to interacting proteins,
and (3) an alkyne handle for reporter tag conjugation to visualize
and identify cross-linked proteins. A library member was found to
inhibit cancer cell proliferation selectively under nutrient-limiting
(low glucose) conditions. Quantitative chemoproteomics identified
MT-ND1, an integral membrane subunit of the ∼1 MDa NADH:ubiquinone
oxidoreductase (complex 1) involved in oxidative phosphorylation,
as a specific target of the active probe. We further demonstrated
that the active probe inhibits complex 1 activity in vitro (IC<sub>50</sub> = 720 nM), an effect that is known to induce cell death
in low-glucose conditions. Based on this proof of principle study,
we anticipate that the generation and integration of fully functionalized
compound libraries into phenotypic screening programs should facilitate
the discovery of bioactive probes that are amenable to accelerated
target identification and mechanistic characterization using advanced
chemoproteomic technologies
Fully Functionalized Small-Molecule Probes for Integrated Phenotypic Screening and Target Identification
Phenotypic screening offers a powerful approach to identify
small
molecules that perturb complex biological processes in cells and organisms.
The tendency of small molecules, however, to interact with multiple
protein targets, often with moderate to weak affinities, along with
the lack of straightforward technologies to characterize these interactions
in living systems, has hindered efforts to understand the mechanistic
basis for pharmacological activity. Here we address this challenge
by creating a fully functionalized small-molecule library whose membership
is endowed with: (1) one or more diversity elements to promote interactions
with different protein targets in cells, (2) a photoreactive group
for UV light-induced covalent cross-linking to interacting proteins,
and (3) an alkyne handle for reporter tag conjugation to visualize
and identify cross-linked proteins. A library member was found to
inhibit cancer cell proliferation selectively under nutrient-limiting
(low glucose) conditions. Quantitative chemoproteomics identified
MT-ND1, an integral membrane subunit of the ∼1 MDa NADH:ubiquinone
oxidoreductase (complex 1) involved in oxidative phosphorylation,
as a specific target of the active probe. We further demonstrated
that the active probe inhibits complex 1 activity in vitro (IC<sub>50</sub> = 720 nM), an effect that is known to induce cell death
in low-glucose conditions. Based on this proof of principle study,
we anticipate that the generation and integration of fully functionalized
compound libraries into phenotypic screening programs should facilitate
the discovery of bioactive probes that are amenable to accelerated
target identification and mechanistic characterization using advanced
chemoproteomic technologies
ABHD4 Regulates Multiple Classes of <i>N</i>‑Acyl Phospholipids in the Mammalian Central Nervous System
<i>N</i>-Acyl phospholipids are atypical components of
cell membranes that bear three acyl chains and serve as potential
biosynthetic precursors for lipid mediators such as endocannabinoids.
Biochemical studies have implicated ABHD4 as a brain <i>N</i>-acyl phosphatidylethanolamine (NAPE) lipase, but <i>in vivo</i> evidence for this functional assignment is lacking. Here, we describe
ABHD4<sup>–/–</sup> mice and their characterization
using untargeted lipidomics to discover that ABHD4 regulates multiple
classes of brain <i>N</i>-acyl phospholipids. In addition
to showing reductions in brain glycerophospho-NAEs (GP-NAEs) and plasmalogen-based
lyso-NAPEs (lyso-pNAPEs), ABHD4<sup>–/–</sup> mice exhibited
decreases in a distinct set of brain lipids that were structurally
characterized as <i>N</i>-acyl lysophosphatidylserines (lyso-NAPSs).
Biochemical assays confirmed that NAPS lipids are direct substrates
of ABHD4. These findings, taken together, designate ABHD4 as a principal
regulator of <i>N</i>-acyl phospholipid metabolism in the
mammalian nervous system
Mapping the Protein Interaction Landscape for Fully Functionalized Small-Molecule Probes in Human Cells
Phenotypic screening provides a means
to discover small molecules
that perturb cell biological processes. Discerning the proteins and
biochemical pathways targeted by screening hits, however, remains
technically challenging. We recently described the use of small molecules
bearing photoreactive groups and latent affinity handles as fully
functionalized probes for integrated phenotypic screening and target
identification. The general utility of such probes, or, for that matter,
any small-molecule screening library, depends on the scope of their
protein interactions in cells, a parameter that remains largely unexplored.
Here, we describe the synthesis of an ∼60-member fully functionalized
probe library, prepared from Ugi-azide condensation reactions to impart
structural diversity and introduce diazirine and alkyne functionalities
for target capture and enrichment, respectively. In-depth mass spectrometry-based
analysis revealed a diverse array of probe targets in human cells,
including enzymes, channels, adaptor and scaffolding proteins, and
proteins of uncharacterized function. For many of these proteins,
ligands have not yet been described. Most of the probe–protein
interactions showed well-defined structure–activity relationships
across the probe library and were blocked by small-molecule competitors
in cells. These findings indicate that fully functionalized small
molecules canvas diverse segments of the human proteome and hold promise
as pharmacological probes of cell biology
Mapping the Protein Interaction Landscape for Fully Functionalized Small-Molecule Probes in Human Cells
Phenotypic screening provides a means
to discover small molecules
that perturb cell biological processes. Discerning the proteins and
biochemical pathways targeted by screening hits, however, remains
technically challenging. We recently described the use of small molecules
bearing photoreactive groups and latent affinity handles as fully
functionalized probes for integrated phenotypic screening and target
identification. The general utility of such probes, or, for that matter,
any small-molecule screening library, depends on the scope of their
protein interactions in cells, a parameter that remains largely unexplored.
Here, we describe the synthesis of an ∼60-member fully functionalized
probe library, prepared from Ugi-azide condensation reactions to impart
structural diversity and introduce diazirine and alkyne functionalities
for target capture and enrichment, respectively. In-depth mass spectrometry-based
analysis revealed a diverse array of probe targets in human cells,
including enzymes, channels, adaptor and scaffolding proteins, and
proteins of uncharacterized function. For many of these proteins,
ligands have not yet been described. Most of the probe–protein
interactions showed well-defined structure–activity relationships
across the probe library and were blocked by small-molecule competitors
in cells. These findings indicate that fully functionalized small
molecules canvas diverse segments of the human proteome and hold promise
as pharmacological probes of cell biology
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Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors
Electrophilic
small molecules are an important class of chemical
probes and drugs that produce biological effects by irreversibly modifying
proteins. Examples of electrophilic drugs include covalent kinase
inhibitors that are used to treat cancer and the multiple sclerosis
drug dimethyl fumarate. Optimized covalent drugs typically inactivate
their protein targets rapidly in cells, but ensuing time-dependent,
off-target protein modification can erode selectivity and diminish
the utility of reactive small molecules as chemical probes and therapeutics.
Here, we describe an approach to confer kinetic selectivity to electrophilic
drugs. We show that an analogue of the covalent Bruton’s tyrosine
kinase (BTK) inhibitor Ibrutinib bearing a fumarate ester electrophile
is vulnerable to enzymatic metabolism on a time-scale that preserves
rapid and sustained BTK inhibition, while thwarting more slowly accumulating
off-target reactivity in cell and animal models. These findings demonstrate
that metabolically labile electrophilic groups can endow covalent
drugs with kinetic selectivity to enable perturbation of proteins
and biochemical pathways with greater precision
Proteome-Wide Reactivity Profiling Identifies Diverse Carbamate Chemotypes Tuned for Serine Hydrolase Inhibition
Serine
hydrolases are one of the largest and most diverse enzyme
classes in Nature. Inhibitors of serine hydrolases are used to treat
many diseases, including obesity, diabetes, cognitive dementia, and
bacterial and viral infections. Nonetheless, the majority of the 200+
serine hydrolases in mammals still lack selective inhibitors for their
functional characterization. We and others have shown that activated
carbamates, through covalent reaction with the conserved serine nucleophile
of serine hydrolases, can serve as useful inhibitors for members of
this enzyme family. The extent to which carbamates, however, cross-react
with other protein classes remains mostly unexplored. Here, we address
this problem by investigating the proteome-wide reactivity of a diverse
set of activated carbamates <i>in vitro</i> and <i>in vivo</i>, using a combination of competitive and click chemistry
(CC)-activity-based protein profiling (ABPP). We identify multiple
classes of carbamates, including <i>O</i>-aryl, <i>O</i>-hexafluoroisopropyl (HFIP), and <i>O</i>-<i>N</i>-hydroxysuccinimidyl (NHS) carbamates that react selectively
with serine hydrolases across entire mouse tissue proteomes <i>in vivo</i>. We exploit the proteome-wide specificity of HFIP
carbamates to create <i>in situ</i> imaging probes for the
endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and α-β
hydrolase-6 (ABHD6). These findings, taken together, designate the
carbamate as a privileged reactive group for serine hydrolases that
can accommodate diverse structural modifications to produce inhibitors
that display exceptional potency and selectivity across the mammalian
proteome
Remodeling Natural Products: Chemistry and Serine Hydrolase Activity of a Rocaglate-Derived β‑Lactone
Flavaglines
are a class of natural products with potent insecticidal
and anticancer activities. β-Lactones are a privileged structural
motif found in both therapeutic agents and chemical probes. Herein,
we report the synthesis, unexpected light-driven di-epimerization,
and activity-based protein profiling of a novel rocaglate-derived
β-lactone. In addition to <i>in vitro</i> inhibition
of the serine hydrolases ABHD10 and ACOT1/2, the most potent β-lactone
enantiomer was also found to inhibit these enzymes, as well as the
serine peptidases CTSA and SCPEP1, in PC3 cells
Remodeling Natural Products: Chemistry and Serine Hydrolase Activity of a Rocaglate-Derived β‑Lactone
Flavaglines
are a class of natural products with potent insecticidal
and anticancer activities. β-Lactones are a privileged structural
motif found in both therapeutic agents and chemical probes. Herein,
we report the synthesis, unexpected light-driven di-epimerization,
and activity-based protein profiling of a novel rocaglate-derived
β-lactone. In addition to <i>in vitro</i> inhibition
of the serine hydrolases ABHD10 and ACOT1/2, the most potent β-lactone
enantiomer was also found to inhibit these enzymes, as well as the
serine peptidases CTSA and SCPEP1, in PC3 cells
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