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Cellular glucose uptake is a key feature reflecting metabolic demand of cells in physiopathological conditions. Fluorophore-conjugated sugar derivatives are widely used for monitoring glucose transporter (GLUT) activity at the single-cell level, but have limitations in in vivo applications. Here, we develop a click chemistry-based post-labeling method for flow cytometric measurement of glucose uptake with low background adsorption. This strategy relies on GLUT-mediated uptake of azide-tagged sugars, and subsequent intracellular labeling with a cell-permeable fluorescent reagent via a copper-free click reaction. Screening a library of azide-substituted monosaccharides, we discover 6-azido-6-deoxy-D-galactose (6AzGal) as a suitable substrate of GLUTs. 6AzGal displays glucose-like physicochemical properties and reproduces in vivo dynamics similar to ¹⁸F-FDG. Combining this method with multi-parametric immunophenotyping, we demonstrate the ability to precisely resolve metabolically-activated cells with various GLUT activities in ex vivo and in vivo models. Overall, this method provides opportunities to dissect the heterogenous metabolic landscape in complex tissue environments.

Brassinosteroid (BR) hormones promote root growth by controlling meristem size and cell elongation, but the mechanism of BR transport remains elusive. A new study shows that BR precursors move via intercellular pores called plasmodesmata to modulate BR cellular levels and their signaling functions.

Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca²⁺ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca²⁺ ions can regulate EGF-stimulated macropinocytosis. We propose the potential for macropinocytosis inhibition through the regulation of a mechanosensitive channel activity using chemical tools.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.

KChIP-3 (potassium channel interacting protein-3), also known as DREAM (Downstream Regulatory Element Antagonist Modulator) or calsenilin, is a multifunctional calcium binding protein that controls the expression level and/or the activity of several proteins related to calcium homeostasis, neuronal excitability and neuronal survival.1 As an auxiliary protein in the plasma membrane, KChIP-3 interacts with, and regulates, among others, the gating of KV4 potassium channels, L- and T-type voltage-dependent calcium channels, NMDA receptors and the transcriptor factor ATF6, which is involved in the unfolding protein response machinery. Considering that altered neuronal calcium homeostasis and the accumulation of poorly folded proteins are common features of many neurodegenerative pathologies, the KChIP-3 modulation could open new avenues for the treatment of different neurodegenerative diseases. However, up to now, only three KChIP-3 binding molecules have been identified. Hence, there is a clear need for the development of chemical tools to modulate and characterize KChIP-3 activity and its interactions. In this communication we report the rational design and synthesis of new KChIP-3 ligands as pharmacological tools to study its protein-protein interaction network, focusing on those interactions involved in neurodegenerative pathologies.

The desire to study biology in situ has been aided by many imaging techniques. Among these, X-ray fluorescence (XRF) mapping permits observation of elemental distributions in a multichannel manner. However, XRF imaging is underused, in part, because of the difficulty in interpreting maps without an underlying cellular ‘blueprint’; this could be supplied using contrast agents. Carbon nanotubes (CNTs) can be filled with a wide range of inorganic materials, and thus can be used as ‘contrast agents’ if biologically absent elements are encapsulated. Here we show that sealed single-walled CNTs filled with lead, barium and even krypton can be produced, and externally decorated with peptides to provide affinity for sub-cellular targets. The agents are able to highlight specific organelles in multiplexed XRF mapping, and are, in principle, a general and versatile tool for this, and other modes of biological imaging.

In this dissertation, I describe the development of bioorthogonal chemical tools toward the study of bacterial pathogens during infection. Bioorthogonal chemistry has enabled the visualization of biomolecules not amenable to fluorescent protein fusion. By feeding an organism an unnatural metabolite bearing a bioorthogonal chemical reporter, the reporter is incorporated into the cellular biopolymer of interest and can be detected by bioorthogonal reaction with an imaging agent. To adapt this approach towards visualizing pathogenic bacteria in an infection setting, my dissertation research focused on two main areas. First, I developed a family of fluorogenic azide probes activated by bioorthogonal click reactions with alkynes. These probes offer significant advantages in imaging studies where it is challenging to wash away unreacted probe, such as within live cells or in live organisms, the settings in which bacterial pathogens reside. Secondly, I developed a family of unnatural D-alanine analogues bearing cyclooctynes, which are incorporated into nascent peptidoglycan in a wide variety of bacterial species and undergo bioorthogonal reactions with azides. In conjunction with the fluorogenic azide probes I developed, these analogues enabled the visualization of cell wall synthesis in an intracellular bacterial pathogen during infection.

In this dissertation, I describe the development of bioorthogonal chemical tools toward the study of bacterial pathogens during infection. Bioorthogonal chemistry has enabled the visualization of biomolecules not amenable to fluorescent protein fusion. By feeding an organism an unnatural metabolite bearing a bioorthogonal chemical reporter, the reporter is incorporated into the cellular biopolymer of interest and can be detected by bioorthogonal reaction with an imaging agent. To adapt this approach towards visualizing pathogenic bacteria in an infection setting, my dissertation research focused on two main areas. First, I developed a family of fluorogenic azide probes activated by bioorthogonal click reactions with alkynes. These probes offer significant advantages in imaging studies where it is challenging to wash away unreacted probe, such as within live cells or in live organisms, the settings in which bacterial pathogens reside. Secondly, I developed a family of unnatural D-alanine analogues bearing cyclooctynes, which are incorporated into nascent peptidoglycan in a wide variety of bacterial species and undergo bioorthogonal reactions with azides. In conjunction with the fluorogenic azide probes I developed, these analogues enabled the visualization of cell wall synthesis in an intracellular bacterial pathogen during infection.

The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.

Notwithstanding their key roles in therapy and as biological probes, 7% of approved drugs are purported to have no known primary target, and up to 18% lack a well-defined mechanism of action. Using a chemoinformatics approach, we sought to "de-orphanize" drugs that lack primary targets. Surprisingly, targets could be easily predicted for many: Whereas these targets were not known to us nor to the common databases, most could be confirmed by literature search, leaving only 13 Food and Drug Administration-approved drugs with unknown targets; the number of drugs without molecular targets likely is far fewer than reported. The number of worldwide drugs without reasonable molecular targets similarly dropped, from 352 (25%) to 44 (4%). Nevertheless, there remained at least seven drugs for which reasonable mechanism-of-action targets were unknown but could be predicted, including the antitussives clemastine, cloperastine, and nepinalone; the antiemetic benzquinamide; the muscle relaxant cyclobenzaprine; the analgesic nefopam; and the immunomodulator lobenzarit. For each, predicted targets were confirmed experimentally, with affinities within their physiological concentration ranges. Turning this question on its head, we next asked which drugs were specific enough to act as chemical probes. Over 100 drugs met the standard criteria for probes, and 40 did so by more stringent criteria. A chemical information approach to drug-target association can guide therapeutic development and reveal applications to probe biology, a focus of much current interest.