Development Of Chemical Tools Research Articles

Spatial Chemoproteomics for Mapping the Active Proteome.

Functional regulation of cell signaling through dynamic changes in protein activity state as well as spatial organization represent two dynamic, complex, and conserved phenomena in biology. Seemingly separate areas of -omics method development have focused on building tools that can detect and quantify protein activity states, as well as map sub-cellular and intercellular protein organization. Integration of these efforts, through the development of chemical tools and platforms that enable detection and quantification of protein functional states with spatial resolution provide opportunities to better understand heterogeneity in the proteome within cell organelles, multi-cellular tissues, and whole organisms. This review provides an overview of and considerations for major classes of chemical proteomic probes and technologies that enable protein activity mapping from sub-cellular compartments to live animals.

Fluorescent Probes for Lipid Membranes: From the Cell Surface to Organelles

Biomembranes are ubiquitous lipid structures that delimit the cell surface and organelles and operate as platforms for a multitude of biomolecular processes. The development of chemical tools─fluorescent probes─for the sensing and imaging of biomembranes is a rapidly growing research direction, stimulated by a high demand from cell biologists and biophysicists. This Account focuses on advances in these smart molecules, providing a voyage from the cell frontier─plasma membranes (PM)─toward intracellular membrane compartments─organelles. General classification of the membrane probes can be based on targeting principles, sensing profile, and optical response. Probes for PM and organelle membranes are designed based on multiple targeting principles: conjugation with natural lipids or synthetic targeting ligands and in situ cell labeling by bio-orthogonal chemistry, conjugation to protein tags, and receptor-ligand interactions. Thus, to obtain membrane probes targeting PM with selectivity to one leaflet, we designed membrane anchor ligands based on a charged group and an alkyl chain. According to the sensing profile, we define basic membrane markers with constant emission and probes for biophysical and chemical sensing. The markers are built from classical fluorophores, exemplified by a series of bright cyanines and BODIPY dyes bearing the PM anchors (MemBright). Membrane probes for biophysical sensing are based on environment-sensitive fluorophores: (1) polarity-sensitive solvatochromic dyes; (2) viscosity-sensitive fluorescent molecular rotors; (3) mechanosensitive fluorescent flippers; and (4) voltage-sensitive electrochromic dyes. Our solvatochromic probes based on Nile Red (NR12S, NR12A, NR4A), Laurdan (Pro12A), and 3-hydroxyflavone (F2N12S) through polarity-sensing can visualize liquid ordered and disordered phases of lipid membranes, sense lipid order and its heterogeneity in cell PM, detect apoptosis, etc. Chemically sensitive probes, combining a dye, membrane-targeting ligand, and molecular recognition unit, enable the detection of pH, ions, redox species, lipids, and proteins at the biomembrane surface. In terms of the optical response profile, we can identify (1) fluorogenic (turn-on) probes, allowing background-free imaging; (2) ratiometric probes, e.g., solvatochromic probes, which enable ratiometric imaging by changing their emission/excitation color; (3) fluorescence lifetime-responsive probes, e.g., fluorescence molecular rotors and flippers, suitable for fluorescence lifetime imaging (FLIM); and (4) switchable probes, important for single-molecule localization microscopy. We showed that combining solvatochromic probes with on-off switching through a reversible binding specifically to cell PM enables the mapping of their biophysical properties with superior resolution. While the majority of efforts have been focused on PM, the probes for cellular organelles, such as endoplasmic reticulum, mitochondria, Golgi apparatus, etc., emerge rapidly. Thus, nontargeted solvatochromic probes can distinguish organelles by the emission color. Targeted solvatochromic probes based on Nile Red revealed unique signatures of polarity and lipid order of individual organelles and their different sensitivities to oxidative or mechanical stress. Lipid droplets, which are membraneless lipidic structures, constitute another interesting organelle target for probing the cell stress. Currently, we stand at the beginning of a long route with big challenges ahead, in particular (1) to achieve superior organelle specificity; (2) to label specific biomembrane leaflets, notably the inner leaflet of PM; (3) to detect lipid organization in a proximity of specific proteins; and (4) to probe biomembranes in tissues and animals.

Roger Yonchien Tsien. 1 February 1952—24 August 2016

Roger Yonchien Tsien displayed precocious childhood talents in chemistry. After graduating from Harvard University in chemistry and physics in 1972, he pursued a PhD programme in the Physiological Laboratory, Cambridge, under the supervison of Professor Richard Adrian (FRS 1977) with Marshal Scholarship support. His thesis ‘The design and use of organic chemical tools in cellular physiology’ won the Gedge Prize and a Comyns Berkeley Research Fellowship by Gonville and Caius College, supporting his postdoctoral work developing Ca 2+ -sensitive electrodes and fluorescent probes measuring cellular [Ca 2+ ]. His novel tetracarboxylate quin-2 readily accessed cells as its subsequently cleavable acetomethoxy ester and signalled in the physiological [Ca 2+ ] range. At Berkeley, further improved ratiometric and strongly fluorescent probes included the Ca 2+ -sensing indo-1, fura-2 and fluo-3 sensors for other strategic ions and molecules that could photolytically uncage key intracellular regulators. At San Diego, design of fluorescent protein complexes built from the cAMP-binding phosphokinase A was followed by Nobel prizewinning work expressing green-fluorescent protein (GFP) and developing spectral GFP variants forming fluorescent resonance energy transfer (FRET) pairs. Their conjugation with further proteins specifically binding particular ions or molecules under study permitted measurement of these by optical methods. Further detector protein complexes such as DsRed, fluorescent at longer wavelengths, were to complement GFP. These interests culminated in the development of chemical tools with potential diagnostic and therapeutic clinical applications through their optical and magnetic resonance signals. His scientific work bequeathed a lasting wealth of chemical tools invaluable for physiological investigation with both current and future applicability.

Development of Rare Bacterial Monosaccharide Analogs for Metabolic Glycan Labeling in Pathogenic Bacteria.

Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents. Thus, the development of chemical tools that label bacterial glycans is a crucial step toward discovering and targeting these biomolecules. Here, we explore the extent to which metabolic glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare deoxy amino d-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-trideoxygalactose and established that these analogs are differentially incorporated into glycan-containing structures in a range of pathogenic and symbiotic bacterial species. Further application of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find utility in treating a variety of infectious diseases with selectivity.

Abstract 1733: Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells

Abstract Background: Heat shock protein 70 family members play an important role in cancer. They are up-regulated in wide variety of tumors and the increased Hsp70 protein expression correlates with metastases, resistance to treatment and poor prognosis. Multiple mechanisms explain cancer cells dependence on Hsp70, such as inhibition of apoptosis by Hsp70, induction of autophagy and control of stability of onco-proteins. These Hsp70 activities are mediated in cancer by its ability to chaperone and interact with a large number of proteins in a cell-specific, context dependent manner. Hypothesis: Reagents that enable the capture of tumor-specific Hsp70 complexes facilitate the identification of context-dependent Hsp70 interactomes. Results: Our laboratory recently reported the identification of a novel allosteric site located in the nucleotide binding domain of Hsp70 (Chem Biol 2013). It has also reported the discovery of ligands that bind to the allosteric pocket of Hsp70, inhibit its function in cancer cells and result in anti-cancer activity (J Med Chem 2013). Structure-activity relationship studies in this ligand series gave insights on the attachment of specific linkers for the design of Hsp70-related chemical tools. Here we present the design of Hsp70-directed reagents and use biochemical and cell-based methods to validate Hsp70-directed affinity purification probes. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins. Significance: The knowledge derived from the use of such reagents will be extremely valuable not only to understand tumor-specific roles of Hsp70 and associated mechanisms but also to develop rational strategies for the clinical implementation of these agents to cancer treatment. They may also provide clues on the altered functional proteome in individual tumors, a quest yet elusive by today's proteomics methods. Citation Format: Anna A. Rodina, Tony Taldone, Yanlong Kang, Pallav Patel, John Koren, Pengrong Yan, Erica DaGama Gomes, Chenghua Yang, Maulik Patel, Liza Shrestha, Stefan Ochiana, Ronnie Maharaj, Alexander Gozman, Marc Cox, Hediye Erdjument-Bromage, Ronald Hendrickson, Leandro Cerchietti, Ari Melnick, Monica Guzman, Gabriela Chiosis. Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2015-1733