Martin J. Lear

Activity-Based Proteome Profiling of Potential Cellular Targets of Orlistat − An FDA-Approved Drug with Anti-Tumor Activities

Orlistat, or tetrahydrolipstatin (THL), is an FDA-approved antiobesity drug with potential antitumor activities. Cellular off-targets and potential side effects of Orlistat in cancer therapies, however, have not been extensively explored thus far. In this study, we report the total of synthesis of THL-like protein-reactive probes, in which extremely conservative modifications (i.e., an alkyne handle) were introduced in the parental THL structure to maintain the native biological properties of Orlistat, while providing the necessary functionality for target identification via the bio-orthogonal click chemistry. With these natural productlike, cell-permeable probes, we were able to demonstrate, for the first time, this chemical proteomic approach is suitable for the identification of previously unknown cellular targets of Orlistat. In addition to the expected fatty acid synthase (FAS), we identified a total of eight new targets, some of which were further validated by experiments including Western blotting, recombinant protein expression, and site-directed mutagenesis. Our findings have important implications in the consideration of Orlistat as a potential anticancer drug at its early stages of development for cancer therapy. Our strategy should be broadly useful for off-target identification against quite a number of existing drugs and/or candidates, which are also covalent modifiers of their biological targets.

A programmed cell death pathway in the malaria parasite Plasmodium falciparum has general features of mammalian apoptosis but is mediated by clan CA cysteine proteases

Several recent discoveries of the hallmark features of programmed cell death (PCD) in Plasmodium falciparum have presented the possibility of revealing novel targets for antimalarial therapy. Using a combination of cell-based assays, flow cytometry and fluorescence microscopy, we detected features including mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in parasites induced with chloroquine (CQ) and staurosporine (ST). The use of the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (zVAD), and the mitochondria outer membrane permeabilization (MOMP) inhibitor, 4-hydroxy-tamoxifen, enabled the characterization of a novel CQ-induced pathway linking cysteine protease activation to downstream mitochondrial dysregulation, amplified protease activity and DNA fragmentation. The PCD features were observed only at high (μM) concentrations of CQ. The use of a new synthetic coumarin-labeled chloroquine (CM-CQ) showed that these features may be associated with concentration-dependent differences in drug localization. By further using cysteine protease inhibitors z-Asp-Glu-Val-Asp-fmk (zDEVD), z-Phe-Ala-fmk (zFA), z-Phe-Phe-fmk (zFF), z-Leu-Leu-Leu-fmk (zLLL), E64d and CA-074, we were able to implicate clan CA cysteine proteases in CQ-mediated PCD. Finally, CQ induction of two CQ-resistant parasite strains, 7G8 and K1, reveals the existence of PCD features in these parasites, the extent of which was less than 3D7. The use of the chemoreversal agent verapamil implicates the parasite digestive vacuole in mediating CQ-induced PCD.

A bismuth(III)-catalyzed Friedel−Crafts cyclization and stereocontrolled organocatalytic approach to (−)-platensimycin

A high yielding route to the (-)-platensimycin core is communicated. This entailed the discovery of Bi(OTf)(3) to catalyze a Friedel-Crafts cyclization of a free lactol, supplemented by LiClO(4) to suppress the Lewis basicity of the sulfonate group. After TBAF-promoted cyclodearomatization, a diastereoselective conjugate reduction of a dienone was achieved by adopting amine-based organocatalytic rationales to reverse the inherent steric control of the substrate.

A Direct and Efficient α-Selective Glycosylation Protocol for the Kedarcidin Sugar, L-Mycarose: AgPF6 as a Remarkable Activator of 2-Deoxythioglycosides

The α-selective effect and potency of AgPF6 on readily prepared 2-deoxythioglycosides allows the direct attachment of a partially protected allo-sugar, such as L-mycarose, to an advanced and hindered aglycon unit of kedarcidin. This method also permits access to other 2-deoxyglycosides in an expedient and mild manner. PMBM=p-methoxybenzyloxymethyl, Alloc=allyloxycarbonyl, TES=triethylsilyl.

Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome

As current diagnostic markers for dry eye syndrome (DES) are lacking in both sensitivity and specificity, a pressing concern exists to develop activity markers that closely align with the principal axes of disease progression. In this study, a comprehensive lipidomic platform designated for analysis of the human tear lipidome was employed to characterize changes in tear lipid compositions from a cohort of 93 subjects of different clinical subgroups classified based on the presence of dry eye symptoms and signs. Positive correlations were observed between the tear levels of cholesteryl sulfates and glycosphingolipids with physiological secretion of tears, which indicated the possible lacrimal (instead of meibomian) origin of these lipids. Notably, we found wax esters of low molecular masses and those containing saturated fatty acyl moieties were specifically reduced with disease and significantly correlated with various DES clinical parameters such as ocular surface disease index, tear breakup time, and Schirmers I test (i.e., both symptoms and signs). These structure-specific changes in tear components with DES could potentially serve as unifying indicators of disease symptoms and signs. In addition, the structurally-specific aberrations in tear lipids reported here were found in patients with or without aqueous deficiency, suggesting a common pathology for both DES subtypes.

Evaluation of aroma-active compounds in Pontianak orange peel oil ( Citrus nobilis Lour. Var. microcarpa Hassk.) by gas chromatography-olfactometry, aroma reconstitution, and omission test.

The aroma-active compounds of Pontianak orange peel oil ( Citrus nobilis Lour. var. microcarpa Hassk.) were characterized by using gas chromatography-olfactometry (GC-O) and aroma extract dilution analysis (AEDA) techniques. Forty-one compounds were found to be aroma-active, which were mainly dominated by saturated and unsaturated aldehydes. The flavor dilution (FD) factor was within the range of 2-2048, and compounds having the highest FD factor were alpha-pinene, beta-pinene, linalool, and 2-methoxy-3-(2-methylpropyl) pyrazine, including a few unknown compounds. On the basis of GC-O results, odor activity value (OAV) and relative flavor activity (RFA) were determined for aroma model reconstitution. These resembled the original aroma of the peel oil for the green, fatty, fresh, peely, floral, and tarry attributes, with the model solution derived from OAV being the closest to Pontianak oil. Omission tests were carried out to verify the significance of (Z)-5-dodecenal and 1-phenylethyl mercaptan as key compounds in the aroma of Pontianak orange peel oil.

Remote 1,6‐Stereocontrol by Iminium‐mediated Organocatalytic Events

How can you catalyse the 1,6-conjugate addition of carbon-nucleophiles to acyclic, achiral 2,4-dienals enantioselectively? This question raises two key issues in catalysis: how to favour 1,6-regioisomers (over 1,4- or 1,2-adducts) and how to achieve a stereodifferentiating event at a weakly electrophilic site. While these issues are challenging enough, this very feat has been achieved by the Jorgensen group under remote iminium-stereocontrol, whereby δ-alkyl dienals can react with olefinic lactones in the presence of the Jorgensen-Hayashi catalyst to give 1,6-adducts exclusively.

Identification of the Binding of Sceptrin to MreB via a Bidirectional Affinity Protocol

A bidirectional affinity system was used to screen for marine natural products that bound to Escherichia coli proteins. A system was developed and applied to isolate the natural product sceptrin from an Agelas conifera extract and its affinity partner MreB from E. coli lysate. The use of a dual immunoaffinity fluorescent (IAF) tag permitted this process to co-immunoprecipitate the bacterial equivalent of actin, MreB, from E. coli lysate. MreB was subsequently validated as a target for sceptrin using a resistance mapping approach. The combination of these studies suggests that natural products and their protein targets can be isolated in concert using a melody of forward and reverse affinity matrices. While the structure of sceptrin was elucidated by NMR analysis, the bulk of effort was conducted without knowing the structure of the natural product, thereby elevating a key bottleneck in the development of high-throughput methods for natural product discovery.

Oxidative Amidation of Nitroalkanes with Amine Nucleophiles using Molecular Oxygen and Iodine

The formation of amides and peptides often necessitates powerful yet mild reagent systems. The reagents used, however, are often expensive and highly elaborate. New atom-economical and practical methods that achieve such goals are highly desirable. Ideally, the methods should start with substrates that are readily available in both chiral and non-chiral forms and utilize cheap reagents that are compatible with a wide variety of functional groups, steric encumberance, and epimerizable stereocenters. A direct oxidative method was developed to form amide and peptide bonds between amines and primary nitroalkanes simply by using I2 and K2 CO3 under O2 . Contrary to expectations, a 1:1 halogen-bonded complex forms between the iodonium source and the amine, which reacts with nitronates to form α-iodo nitroalkanes as precursors to the amides.

Chemical Modification and Organelle‐Specific Localization of Orlistat‐Like Natural‐Product‐Based Probes

Orlistat, also known as tetrahydrolipstatin (THL), is an FDA-approved anti-obesity drug with potential anti-cancer activity. Previously, we developed a chemical proteomic approach, based on the Orlistat-like probe (1a) for large-scale identification of unknown cellular targets of Orlistat in human hepatocytes. In this article, we report the chemical synthesis and biological evaluation of an expanded set of Orlistat-like compounds, with the intention to further dissect and manipulate potential cellular targets of Orlistat. In doing so, we carried out proteome-wide activity-based profiling and large-scale pull-down/LCMS analysis of these compounds in live HepG2 cells, and successfully identified many putative cellular targets for Orlistat and its structural analogues. By qualitatively assessing the spectra counts of potential protein hits against each of the seventeen Orlistat analogues, we obtained both common and unique targets of these probes. Our results revealed that subtle structural modifications of Orlistat led to noticeable changes in both the cellular potency and target profiles of the drug. In order to further improve the cellular activity of Orlistat, we successfully applied the well-established AGT/SNAP-tag technology to our cell-permeable, benzylguanine (BG)-containing Orlistat variant (4). We showed that the drug could be delivered and effectively retained in different sub-cellular organelles of living cells. This strategy may provide a general and highly effective chemical tool for the potential sub-cellular targeting of small molecule drugs.