Micah J. Niphakis

Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells

Cholesterol is an essential structural component of cellular membranes and serves as a precursor for several classes of signaling molecules. Cholesterol exerts its effects and is, itself, regulated in large part by engagement in specific interactions with proteins. The full complement of sterol-binding proteins that exist in mammalian cells, however, remains unknown. Here we describe a chemoproteomic strategy that uses clickable, photoreactive sterol probes in combination with quantitative mass spectrometry to globally map cholesterol-protein interactions directly in living cells. We identified over 250 cholesterol-binding proteins, including receptors, channels and enzymes involved in many established and previously unreported interactions. Prominent among the newly identified interacting proteins were enzymes that regulate sugars, glycerolipids and cholesterol itself as well as proteins involved in vesicular transport and protein glycosylation and degradation, pointing to key nodes in biochemical pathways that may couple sterol concentrations to the control of other metabolites and protein localization and modification.

Palladium(II)-Catalyzed Direct Arylation of Enaminones Using Organotrifluoroborates

A Pd(II)-catalyzed reaction for the direct arylation of cyclic enaminones is reported. The reactivity of electron-rich, electron-poor, and sterically encumbered organotrifluoroborates was investigated. This reaction represents a unique use for organotrifluoroborates as coupling partners and discloses the utility of enaminones for direct-functionalization reactions. It provides immediate access to arylpiperidine, indolizidine, and quinolizidine scaffolds from the corresponding mono- and bicyclic, unattenuated enaminones.

Enzyme Inhibitor Discovery by Activity-Based Protein Profiling

Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.

Determining target engagement in living systems

Chemical probes are critical tools for elucidating the biological functions of proteins and can lead to new medicines for treating disease. The pharmacological validation of protein function requires verification that chemical probes engage their intended targets in vivo. Here we discuss technologies, both established and emergent, for measuring target engagement in living systems and propose that determining this parameter should become standard practice for chemical probe and drug discovery programs.

Highly Selective Inhibitors of Monoacylglycerol Lipase Bearing a Reactive Group that Is Bioisosteric with Endocannabinoid Substrates

The endocannabinoids 2-arachidonoyl glycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide) are principally degraded by monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively. The recent discovery of O-aryl carbamates such as JZL184 as selective MAGL inhibitors has enabled functional investigation of 2-AG signaling pathways in vivo. Nonetheless, JZL184 and other reported MAGL inhibitors still display low-level cross-reactivity with FAAH and peripheral carboxylesterases, which can complicate their use in certain biological studies. Here, we report a distinct class of O-hexafluoroisopropyl (HFIP) carbamates that inhibits MAGL in vitro and in vivo with excellent potency and greatly improved selectivity, including showing no detectable cross-reactivity with FAAH. These findings designate HFIP carbamates as a versatile chemotype for inhibiting MAGL and should encourage the pursuit of other serine hydrolase inhibitors that bear reactive groups resembling the structures of natural substrates.

Palladium(II)-catalyzed dehydrogenative alkenylation of cyclic enaminones via the Fujiwara-Moritani reaction

A new Pd(II)-catalyzed dehydrogenative alkenylation reaction involving two alkenes was developed. A variety of nonaromatic, cyclic enaminones were successfully coupled to primary and secondary alkenes yielding a series of unique 1,3-dienes. The generality of this transformation presents a useful strategy for directly cross-coupling alkenes and offers an attractive new approach to functionalize enaminones.

In vivo characterization of the highly selective monoacylglycerol lipase inhibitor KML29: Antinociceptive activity without cannabimimetic side effects:

Since monoacylglycerol lipase (MAGL) has been firmly established as the predominant catabolic enzyme of the endocannabinoid 2‐arachidonoylglycerol (2‐AG), a great need has emerged for the development of highly selective MAGL inhibitors. Here, we tested the in vivo effects of one such compound, KML29 (1,1,1,3,3,3‐hexafluoropropan‐2‐yl 4‐(bis(benzo[d][1,3]dioxol‐5‐yl)(hydroxy)methyl)piperidine‐1‐carboxylate).

A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells

Lipids play central roles in physiology and disease, where their structural, metabolic, and signaling functions often arise from interactions with proteins. Here, we describe a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells. These interactions involve hundreds of proteins from diverse functional classes and frequently occur at sites of drug action. We determine the target profiles for several drugs across the lipid-interaction proteome, revealing that its ligandable content extends far beyond traditionally defined categories of druggable proteins. In further support of this finding, we describe a selective ligand for the lipid-binding protein nucleobindin-1 (NUCB1) and show that this compound perturbs the hydrolytic and oxidative metabolism of endocannabinoids in cells. The described chemical proteomic platform thus provides an integrated path to both discover and pharmacologically characterize a wide range of proteins that participate in lipid pathways in cells.

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 in vitro and in vivo, using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ 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.

Synthesis of tylocrebrine and related phenanthroindolizidines by VOF3-mediated oxidative aryl-alkene coupling.

A highly convergent strategy to prepare phenanthroindolizidines is reported involving three consecutive C-C coupling reactions. This sequence features a novel VOF(3)-mediated aryl-alkene coupling in the final step, which enables regioselective preparation of C5-substituted phenanthroindolizidines for the first time. This strategy has been applied to the synthesis of eight natural and unnatural members in this class to investigate the scope of this chemistry and to explore structure-activity relationships.