Copper-Assisted Palladium(II)-Catalyzed Direct Arylation of Cyclic Enaminones with Arylboronic Acids

Described herein is a palladium­(II)-catalyzed direct arylation of cyclic enaminones with arylboronic acids. The versatility of this method is that both electron-rich and electron-poor boronic acids can be coupled in high yields. A mixture of two Cu­(II) additives was crucial for efficient cross-coupling. The role of each Cu­(II) reagent appears to be distinct and complementary serving to assist catalyst reoxidation and transmetalation through a putative arylcopper intermediate

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

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

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

Evaluation of NHS Carbamates as a Potent and Selective Class of Endocannabinoid Hydrolase Inhibitors

Monoacylglycerol lipase (MAGL) is a principal metabolic enzyme responsible for hydrolyzing the endogenous cannabinoid (endocannabinoid) 2-arachidonoylglycerol (2-AG). Selective inhibitors of MAGL offer valuable probes to further understand the enzyme’s function in biological systems and may lead to drugs for treating a variety of diseases, including psychiatric disorders, neuroinflammation, and pain. <i>N</i>-Hydroxysuccinimidyl (NHS) carbamates have recently been identified as a promising class of serine hydrolase inhibitors that shows minimal cross-reactivity with other proteins in the proteome. Here, we explore NHS carbamates more broadly and demonstrate their potential as inhibitors of endocannabinoid hydrolases and additional enzymes from the serine hydrolase class. We extensively characterize an NHS carbamate <b>1a</b> (MJN110) as a potent, selective, and in-vivo-active MAGL inhibitor. Finally, we demonstrate that MJN110 alleviates mechanical allodynia in a rat model of diabetic neuropathy, marking NHS carbamates as a promising class of MAGL inhibitors