ABHD17 regulation of plasma membrane palmitoylation and N-Ras-dependent cancer growth.

Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.

1,6-Dioxo-2-azaspiro[3.4]oct-2-enes and Related Spirocycles: Heterocycles from [3 + 2] Nitrile Oxide Cycloadditions with 2-Methyleneoxetanes, -Thietanes, and -Azetidines.

Isoxazolines and 4-membered heterocycles are significant structural motifs in numerous synthetic intermediates and natural products. [3 + 2] Cycloadditions between enol ethers and nitrile oxides have been well studied; however, nitrile oxide cycloadditions with 4-membered heterocycles to give spiroisoxazolines are unreported. Here, we showcase the regio- and diastereoselective [3 + 2] nitrile oxide cycloadditions of 2-methyleneoxetanes, -azetidines, and -thietanes to give an array of 1,6-dioxo-2-azaspiro[3.4]oct-2-enes and related spirocycles. 2D NMR experiments suggested that most of the observed diastereoselectivities were dictated by steric interactions; however, dipolarophiles with H bonding donors reversed the stereochemical outcome. X-ray crystallography confirmed the structural assignments.

Unusual Transformations of Strain-Heightened Oxetanes.

Oxetanes are important motifs for drug discovery and are valuable templates in organic synthesis. Much of their use as synthetic intermediates exploits their inherent strain, often resulting in chain extensions at the expense of the heterocycle. Modifications on the carbon alpha to the oxygen of oxetanes, such as the C═O of ß-lactones, extend the modes of reactivity. Nevertheless, the outcomes are still largely predictable. On the other hand, other alpha modifications, such as a ═CH2, a spiro-oxiranyl moiety, or a spiro-cyclopropyl group, increase strain and open pathways not available to simple oxetanes or ß-lactones. Methods in generating 2-methyleneoxetanes, 1,5-dioxaspiro[3.2]hexanes, and 4-oxaspiro[2.3]hexanes have been developed by us and others. To date, reactions of these systems have sometimes been predictable, but often the outcomes have been unexpected. This has provided fertile ground for thinking about what controls reactivity and what other reaction pathways might be accessible to these strain-heightened oxetanes.This Account summarizes the published literature on the most straightforward approaches to 2-methyleneoxetanes, dioxaspirohexanes, and oxaspirohexanes and on their reactivity. In contrast to simple oxetanes, reactions of 2-methyleneoxetanes with nucleophiles at C4 release an enolate rather than an alkoxide. Also, 2-methyleneoxetanes can be converted to homopropargyl alcohols or undergo a silicon accelerated isomerization/electrocyclic ring opening, processes accessible only because of the exocyclic double bond. In addition, oxetane oxocarbenium ions, derived from protonation of the enol ether, can react with nucleophiles to provide 2,2-disubstituted oxetanes. Oxaspirohexanes are readily prepared by Simmons-Smith cyclopropanation of 2-methyleneoxetanes. These unusual systems undergo a variety of substituent dependent rearrangements in the presence of the Lewis acid BF3·Et2O. In addition, upon treatment with Zeise's dimer, oxaspirohexanes are transformed to synthetically useful 3-methylenetetrahydrofurans. Dioxaspirohexanes are easily accessed by dimethyldioxirane oxidation of 2-methyleneoxetanes. Predictably, dioxaspirohexanes react with many nucleophiles to give α-functionalized-ß'-hydroxy ketones. Unexpectedly, 2,2-disubstituted oxetanes can also be selectively produced. This latter pathway has led to further unusual transformations, illuminating computational studies, and novel routes to biologically relevant molecules.

α-Methylene-ß-Lactone Scaffold for Developing Chemical Probes at the Two Ends of the Selectivity Spectrum.

The utilities of an α-methylene-ß-lactone (MeLac) moiety as a warhead composed of multiple electrophilic sites are reported. We demonstrate that a MeLac-alkyne not only reacts with diverse proteins as a broadly reactive measurement probe, but also recruits reduced endogenous glutathione (GSH) to assemble a selective chemical probe of GSH-ß-lactone (GSH-Lac)-alkyne in live cells. Tandem mass spectrometry reveals that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael or acyl addition. A peptide-centric proteomics platform demonstrates that the proteomic selectivity profiles of orlistat and parthenolide, which have distinct reactivities, are measurable by MeLac-alkyne as a high-coverage probe. The GSH-Lac-alkyne selectively probes the glutathione S-transferase P responsible for multidrug resistance. The assembly of the GSH-Lac probe exemplifies a modular and scalable route to develop selective probes with different recognizing moieties.

Chemical proteomic analysis of palmostatin beta-lactone analogs that affect N-Ras palmitoylation.

S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.

A molecular basis for the exquisite CD1d-restricted antigen specificity and functional responses of natural killer T cells.

Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.

Contact sensitizers trigger human CD1-autoreactive T-cell responses.

Allergic contact dermatitis is a primarily T-cell-mediated inflammatory skin disease induced by exposure to small molecular-weight haptens, which covalently bind to proteins. The abundance of cutaneous T cells that recognize CD1a antigen-presenting molecules raises the possibility that MHC-independent antigen presentation may be relevant in some hapten-driven immune responses. Here we examine the ability of contact sensitizers to influence CD1-restricted immunity. Exposure of human antigen-presenting cells such as monocyte-derived dendritic cells and THP-1 cells to the prototypical contact sensitizer dinitrochlorobenzene potentiated the response of CD1a- and CD1d-autoreactive T cells, which released a vast array of cytokines in a CD1- and TCR-dependent manner. The potentiating effects of dinitrochlorobenzene depended upon newly synthesized CD1 molecules and the presence of endogenous stimulatory lipids. Further examination of a broad panel of contact sensitizers revealed 1,4-benzoquinone, resorcinol, isoeugenol, and cinnamaldehyde to activate the same type of CD1-restricted responses. These findings provide a basis for the antigen-specific activation of skin-associated CD1-restricted T cells by small molecules and may have implications for contact sensitizer-induced inflammatory skin diseases.

T cell receptor CDR2 beta and CDR3 beta loops collaborate functionally to shape the iNKT cell repertoire.

Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.

Kinetics and cellular site of glycolipid loading control the outcome of natural killer T cell activation.

CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

Scaling Proteome-Wide Reactions of Activity-Based Probes.

Unified analysis of complex reactions of an activity-based probe with proteins in a proteome remains an unsolved challenge. We propose a power expression, rate = kobs[Probe]α, for scaling the progress of proteome-wide reactions and use the scaling factor (0 ≤ α ≤ 1) as an apparent, partial order with respect to the probe to measure the "enzyme-likeness" for a protein in reaction acceleration. Thus, α reports the intrinsic reactivity of the protein with the probe. When α = 0, the involved protein expedites the reaction to the maximal degree; when α = 1, the protein reacts with the probe via an unaccelerated, bimolecular reaction. The selectivity (ß) of the probe reacting with two proteins is calculated as a ratio of conversion factors (kobs values) for corresponding power equations. A combination of α and ß provides a tiered system for quantitatively assessing the probe efficacy; an ideal probe exhibits high reactivity with its protein targets (low in α) and is highly selective (high in ß) in forming the probe-protein adducts. The scaling analysis was demonstrated using proteome-wide reactions of HT-29 cell lysates with a model probe of threonine ß-lactone.

Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay.

Lysophosphatidylserines (lyso-PSs) are a class of signaling lipids that regulate immunological and neurological processes. The metabolism of lyso-PSs remains poorly understood in vivo. Recently, we determined that ABHD12 is a major brain lyso-PS lipase, implicating lyso-PSs in the neurological disease polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC), which is caused by null mutations in the ABHD12 gene. Here, we couple activity-based profiling with pharmacological and genetic methods to annotate the poorly characterized enzyme ABHD16A as a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. We describe a small-molecule inhibitor of ABHD16A that depletes lyso-PSs from cells, including lymphoblasts derived from subjects with PHARC. In mouse macrophages, disruption of ABHD12 and ABHD16A respectively increases and decreases both lyso-PSs and lipopolysaccharide-induced cytokine production. Finally, Abhd16a(-/-) mice have decreased brain lyso-PSs, which runs counter to the elevation in lyso-PS in Abhd12(-/-) mice. Our findings illuminate an ABHD16A-ABHD12 axis that dynamically regulates lyso-PS metabolism in vivo, designating these enzymes as potential targets for treating neuroimmunological disorders.

1,4-Dicarbofunctionalization of 4-Fluoroaryl Grignard and Lithium Reagents with Disubstituted Malononitriles.

An efficient one-pot 1,4-dicarbofunctionalization of 4-fluoroaryl Grignard or lithium reagents with 2,2-disubstituted malononitriles is described. The reaction proceeds by sequential transnitrilation and SNAr reactions. Commercial Grignard solutions, Grignard reagents prepared in situ by halogen/magnesium exchange with i-PrMgCl, or aryllithium reagents prepared in situ by bromine/lithium exchange with n-BuLi are compatible with the reaction conditions. Moreover, 2,2-disubstituted malononitriles of diverse structures are accommodated. The reaction provides a unique approach to 1,4-dicarbofunctionalization of activated arenes in a tandem, one-pot transformation.

Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.

Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness.

Rhodium-Catalyzed Addition of Aryl Boronic Acids to 2,2-Disubstituted Malononitriles.

ß-Ketonitriles bearing a quaternary carbon at the 2-position were prepared through Rh-catalyzed addition of aryl boronic acids to 2,2-disubstituted malononitriles. In contrast to the previously described transnitrilative cyanation of aryl boronic acids with dialkylmalononitriles, the present reaction avoids retro-Thorpe collapse of the intermediate addition product through the use of a milder base. The reaction was amenable to a variety of aryl boronic acids and disubstituted malononitriles, providing a diverse array of ß-ketonitriles. The products could be further derivatized to valuable chiral α,α-disubstituted-ß-aminonitriles through addition reactions to the corresponding N-tert-butanesulfinyl imines.

The Alpha and Omega of Galactosylceramides in T Cell Immune Function.

Glycosphingolipids are a subgroup of glycolipids that contain an amino alcohol sphingoid base linked to sugars. They are found in the membranes of cells ranging from bacteria to vertebrates. This group of lipids is known to stimulate the immune system through activation of a type of white blood cell known as natural killer T cell (NKT cell). Here we summarize the extensive research that has been done to identify the structures of natural glycolipids that stimulate NKT cells and to determine how these antigens are recognized. We also review studies designed to understand how glycolipid variants, both natural and synthetic, can alter the responses of NKT cells, leading to dramatic changes in the global immune response.

Rhodium-Catalyzed Transnitrilation of Aryl Boronic Acids with Dimethylmalononitrile.

An efficient transnitrilation of aryl boronic acids with dimethylmalononitrile (DMMN) is described. This rhodium-catalyzed electrophilic cyanation presents a novel approach to prepare aryl nitriles by using a carbon-bound cyanating reagent which undergoes cross-coupling with the aryl boronic acid. The reaction expands the degree of functional-group compatibility exhibited by the transnitrilation of aryl Grignard and aryllithium reagents. A variety of aryl boronic acid derivatives and dialkylmalononitriles were amenable to the transnitrilation.

Recent Applications of Oxetanes in the Synthesis of Heterocyclic Compounds.

Oxetanes are valuable intermediates in organic synthesis, and strategic manipulations of this strained heterocycle continue to emerge. In this Synopsis, recent, distinct approaches to construct heterocyclic systems using oxetanes are described. These include ring expansion, ring opening, and C-2 functionalization.

Pt-catalyzed rearrangement of oxaspirohexanes to 3-methylenetetrahydrofurans: scope and mechanism.

A novel Pt-catalyzed rearrangement of oxaspirohexanes to 3-methylenetetrahydrofurans is reported. Mechanistic studies by (13)C-labeling experiments confirm oxidative addition of Pt(II) regioselectively to the least substituted carbon-carbon bond of the cyclopropane to form a platinacyclobutane intermediate. To our knowledge, this is the first alkoxy-substituted platinacyclobutane that has been observed spectroscopically. The scope and a proposed mechanism of this new Pt-catalyzed transformation are described.

Combining cross-metathesis and activity-based protein profiling: new ß-lactone motifs for targeting serine hydrolases.

ß-Lactones are a privileged structural motif as enzyme inhibitors and chemical probes, particularly for the inhibition of enzymes from the serine hydrolase class. Herein, we demonstrate that cross-metathesis (CM) of α-methylene-ß-lactones offers rapid access to structurally diverse, previously unexplored ß-lactones. Combining this approach with competitive activity-based protein profiling (ABPP) identified lead ß-lactone inhibitors/probes for several serine hydrolases, including disease-associated enzymes and enzymes of uncharacterized function. The structural diversity afforded by the α-methylene-ß-lactone scaffold thus expands the landscape of serine hydrolases that can be targeted by small-molecule inhibitors and should further the functional characterization of enzymes from this class through the optimization of target-selective probes.

A Solvent-free Approach to Glycosyl Amides: Towards the Synthesis of α-N-Galactosyl Ceramides.

A new, simple and efficient method for the synthesis of both α- and ß-glycosyl amides using solvent-free conditions is described. This method involves the coupling of glycosyl amines with the p-nitrophenol esters of lipids as a key step.