A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells.

Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure-activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.

Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer.

In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding ß-galactosylceramide (ßGalCer) without sulfate. C24:2 induced IFN-γ-dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid's function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.

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.

A humanized mouse model for in vivo evaluation of invariant Natural Killer T cell responses.

Invariant natural killer T (iNKT) cells mediate immune responses when stimulated by glycolipid agonists presented by CD1d. In extensive studies of synthetic analogues of α-galactosyl ceramides, we identified numerous examples of significant differences in the recognition of specific glycolipids in wild type mice versus human iNKT cell clones or PBMC samples. To predict human iNKT cell responses more accurately in a mouse model, we derived a mouse line in which compound genetic modifications were used to express a human-like iNKT cell TCR along with human CD1d in place of the endogenous mouse proteins. Detailed transcriptional and phenotypic profiling demonstrated that these partially humanized mice developed an expanded population of T cells recognizing CD1d-presented glycolipid antigens, among which a subset characterized by expression of chemokine receptor CXCR6 had features characteristic of authentic iNKT cells. Responses to iNKT cell activating glycolipids in these mice generated cytokine production in vitro and in vivo that showed a pattern of fine specificity that closely resembled that of cultured human iNKT cell clones. Anti-tumor responses to variants of α-galactosyl ceramide in VαKI mice also correlated with their potency for stimulating human iNKT cells. This genetically modified mouse line provides a practical model for human presentation and recognition of iNKT cell activators in the context of a normally functioning immune system, and may furnish valuable opportunities for preclinical evaluation of iNKT cell-based therapies.

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.

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.

Easily accessible non-aromatic heterocycles with handles: 4-bromo-2,3-dihydrofurans from 1,2-dibromohomoallylic alcohols.

The first general preparation of 4-bromo-2,3-dihydrofurans is reported. These non-aromatic heterocycles containing a useful coupling handle are accessed via Cu-catalyzed intramolecular cyclization of 1,2-dibromohomoallylic alcohols, which are themselves available in just two steps from aromatic and aliphatic aldehydes and ketones. Molecular dynamics simulations using the simple substrates and key geometric parameters provide a rationale for the selectivities observed. The synthetic utility of the 4-bromodihydrofurans is also demonstrated.

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.

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.

Amide-Linked C4″-Saccharide Modification of KRN7000 Provides Potent Stimulation of Human Invariant NKT Cells and Anti-Tumor Immunity in a Humanized Mouse Model.

Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses. In silico analysis suggested that the tether length and degree of flexibility of the amide substituent affected the recognition by iNKT cell antigen receptors of the C4″-amide substituted glycolipids in complex with their antigen presenting molecule CD1d. Our findings establish the use of stable C4″-amide linked additions to the sugar moiety for further exploration of the immunological effects of structural modifications of iNKT cell activating glycolipids and highlight the critical need for more accurate animal models to assess these compounds for immunotherapeutic potential in humans.

Immunomodulatory sphingosine-1-phosphates as plasma biomarkers of Alzheimer's disease and vascular cognitive impairment.

BACKGROUND: There has been ongoing research impetus to uncover novel blood-based diagnostic and prognostic biomarkers for Alzheimer's disease (AD), vascular dementia (VaD), and related cerebrovascular disease (CEVD)-associated conditions within the spectrum of vascular cognitive impairment (VCI). Sphingosine-1-phosphates (S1Ps) are signaling lipids which act on the S1PR family of cognate G-protein-coupled receptors and have been shown to modulate neuroinflammation, a process known to be involved in both neurodegenerative and cerebrovascular diseases. However, the status of peripheral S1P in AD and VCI is at present unclear. METHODS: We obtained baseline bloods from individuals recruited into an ongoing longitudinal cohort study who had normal cognition (N = 80); cognitive impairment, no dementia (N = 160); AD (N = 113); or VaD (N = 31), along with neuroimaging assessments of cerebrovascular diseases. Plasma samples were processed for the measurements of major S1P species: d16:1, d17:1, d18:0, and d18:1, along with pro-inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF). Furthermore, in vitro effects of S1Ps on cytokine expression were also studied in an astrocytoma cell line and in rodent primary astrocytes. RESULTS: Of the S1Ps species measured, only d16:1 S1P was significantly reduced in the plasma of VaD, but not AD, patients, while the d18:1 to d16:1 ratios were increased in all cognitive subgroups (CIND, AD, and VaD). Furthermore, d18:1 to d16:1 ratios correlated with levels of IL-6, IL-8, and TNF. In both primary astrocytes and an astroglial cell line, treatment with d16:1 or d18:1 S1P resulted in the upregulation of mRNA transcripts of pro-inflammatory cytokines, with d18:1 showing a stronger effect than d16:1. Interestingly, co-treatment assays showed that the addition of d16:1 reduced the extent of d18:1-mediated gene expression, indicating that d16:1 may function to "fine-tune" the pro-inflammatory effects of d18:1. CONCLUSION: Taken together, our data suggest that plasma d16:1 S1P may be useful as a diagnostic marker for VCI, while the d18:1 to d16:1 S1P ratio is an index of dysregulated S1P-mediated immunomodulation leading to chronic inflammation-associated neurodegeneration and cerebrovascular damage.

A single-domain bispecific antibody targeting CD1d and the NKT T-cell receptor induces a potent antitumor response.

Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.

Mrp1 is involved in lipid presentation and iNKT cell activation by Streptococcus pneumoniae.

Invariant natural killer T cells (iNKT cells) are activated by lipid antigens presented by CD1d, but the pathway leading to lipid antigen presentation remains incompletely characterized. Here we show a whole-genome siRNA screen to elucidate the CD1d presentation pathway. A majority of gene knockdowns that diminish antigen presentation reduced formation of glycolipid-CD1d complexes on the cell surface, including members of the HOPS and ESCRT complexes, genes affecting cytoskeletal rearrangement, and ABC family transporters. We validated the role in vivo for the multidrug resistance protein 1 (Mrp1) in CD1d antigen presentation. Mrp1 deficiency reduces surface clustering of CD1d, which decreased iNKT cell activation. Infected Mrp1 knockout mice show decreased iNKT cell responses to antigens from Streptococcus pneumoniae and were associated with increased mortality. Our results highlight the unique cellular events involved in lipid antigen presentation and show how modification of this pathway can lead to lethal infection.

Heterogeneous Catalytic Oxidation of Amides to Imides by Manganese Oxides.

Herein, we report a one-step peroxide mediated heterogeneous catalytic oxidation of amides to imides utilizing a series of manganese oxides. Among them, Cs/Mn2O3 was found to be the most active catalyst for the selective partial oxidation of N-benzylbenzamide to diphenyl imide. We have been able to apply an optimized oxidation method to other aromatic substrates. The feasibility of using air as an oxidant, the heterogeneous nature, inexpensive catalytic materials, respectable turnover numbers, and chemoselectivity to imides make this methodology an attractive choice for functional group transformations of amides to imides.

Dual Modifications of α-Galactosylceramide Synergize to Promote Activation of Human Invariant Natural Killer T Cells and Stimulate Anti-tumor Immunity.

Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α-galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly proinflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here, we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α-galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6″ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings and suggested basic principles for capturing useful properties of sphinganine analogs of synthetic iNKT cell activators in the design of immunotherapeutic agents.

Rh-Catalyzed Conjugate Addition of Aryl and Alkenyl Boronic Acids to α-Methylene-ß-lactones: Stereoselective Synthesis of trans-3,4-Disubstituted ß-Lactones.

A one-step preparation of 3,4-disubstituted ß-lactones through Rh-catalyzed conjugate addition of aryl or alkenyl boronic acids to α-methylene-ß-lactones is described. The operationally simple, stereoselective transformation provides a broad range of ß-lactones from individual α-methylene-ß-lactone templates. This methodology allowed for a direct, final-step C-3 diversification of nocardiolactone, an antimicrobial natural product.

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.

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.

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.