Galectin-3-Binding Glycomimetics that Strongly Reduce Bleomycin-Induced Lung Fibrosis and Modulate Intracellular Glycan Recognition.

Discovery of glycan-competitive galectin-3-binding compounds that attenuate lung fibrosis in a murine model and that block intracellular galectin-3 accumulation at damaged vesicles, hence revealing galectin-3-glycan interactions involved in fibrosis progression and in intracellular galectin-3 activities, is reported. 3,3'-Bis-(4-aryltriazol-1-yl)thiodigalactosides were synthesized and evaluated as antagonists of galectin-1, -2, -3, and -4 N-terminal, -4 C-terminal, -7 and -8 N-terminal, -9 N-terminal, and -9 C-terminal domains. Compounds displaying low-nanomolar affinities for galectins-1 and -3 were identified in a competitive fluorescence anisotropy assay. X-ray structural analysis of selected compounds in complex with galectin-3, together with galectin-3 mutant binding experiments, revealed that both the aryltriazolyl moieties and fluoro substituents on the compounds are involved in key interactions responsible for exceptional affinities towards galectin-3. The most potent galectin-3 antagonist was demonstrated to act in an assay monitoring galectin-3 accumulation upon amitriptyline-induced vesicle damage, visualizing a biochemically/medically relevant intracellular lectin-carbohydrate binding event and that it can be blocked by a small molecule. The same antagonist administered intratracheally attenuated bleomycin-induced pulmonary fibrosis in a mouse model with a dose/response profile comparing favorably with that of oral administration of the marketed antifibrotic compound pirfenidone.

Specificity of Lipoprotein Chaperones for the Characteristic Lipidated Structural Motifs of their Cognate Lipoproteins.

Lipoprotein-binding chaperones mediate intracellular transport of lipidated proteins and determine their proper localisation and functioning. Understanding of the exact structural parameters that determine recognition and transport by different chaperones is of major interest. We have synthesised several lipid-modified peptides, representative of different lipoprotein classes, and have investigated their binding to the relevant chaperones PDEδ, UNC119a, UNC119b, and galectins-1 and -3. Our results demonstrate that PDEδ recognises S-isoprenylated C-terminal peptidic structures but not N-myristoylated peptides. In contrast, UNC119 proteins bind only mono-N-myristoylated, but do not recognise doubly lipidated and S-isoprenylated peptides at the C terminus. For galectins-1 and -3, neither binding to N-acylated, nor to C-terminally prenylated peptides could be determined. These results shed light on the specificity of the chaperone-mediated cellular lipoprotein transport systems.

Biology-oriented synthesis: harnessing the power of evolution.

For scientists to gain a better understanding of nature, biological research is greatly aided by small-molecule modulators that perturb protein activity without fundamentally altering the underlying biological systems. The number of possible interfering molecules, however, is so vast that, due to limitations in existing matter and time required for synthesis, they cannot be covered comprehensively. Because proteins and their cognate natural product ligands and substrates co-evolved, these naturally occurring ligands can serve as structural starting points to explore the biologically relevant chemical space. To this end, known natural products are structurally classified on the basis of their core scaffolds and hierarchically arranged in the "natural product tree", which can be annotated for bioactivity and intuitively navigated with currently available software. Biologically relevant scaffolds inspire the synthesis of compound libraries enriched in biological activity. This Perspective describes the development of "biology-oriented synthesis" as a guiding principle to harness the power of evolution in the quest for novel bioactive small molecules for chemical biology research and drug discovery.

Development of a microarray detection method for galectin cancer proteins based on ligand binding.

In this article, we describe the development of a novel detection method for the visualization of ligand-binding proteins. Current proteomic tools, such as the enzyme-linked immunosorbent assay (ELISA), are based on protein abundance rather than protein activity and can result in conflicting data. To address this issue, we developed an assay in which ligand binding is detected using a microarray approach with immobilized antibodies on a porous aluminum oxide matrix. The galectin family of proteins was used as a model system to evaluate the performance of this approach. Galectins selectively bind galactosides and are linked to cancer progression. Our assay employed antibodies directed against different galectins. The antibodies were immobilized on the microarray surface by use of protein A/G. In our example, galectin-1 and galectin-9 were then detected in cell lysates. Lysates were exposed to the anti-galectin surface, followed by washing and quantification with a general fluorescent galectin ligand. The optimal galectin ligand allowed detection of nanogram amounts of galectin using only 1 µg of antibody. Galectin-1 was visualized in HeLa and tumor cell lysates, indicating the potential of the method for a clinical setting.

Functional assay for shiga-like toxin via detection by antibody capture and multivalent galabiose binding.

A functional detection assay was developed for Escherichia coli secreted shiga-like toxin based on antibody capture and visualization with a multivalent galabiose ligand. It was possible to detect verotoxin in medically relevant E. coli samples in a dose dependent fashion. This method is a new step towards measuring functional protein levels in complex mixtures, which can be used for diagnostic purposes in a clinical setting.

The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides.

Hexafluoroisopropanol (HFIP) has been widely used as an acidic modifier for mobile phases for liquid chromatography-mass spectrometry (LC-MS) analysis of oligonucleotides ever since the first report of its use for this purpose. This is not surprising, considering the exceptional performance of HFIP compared with carboxylic acids, which cause significant MS signal suppression in electrospray ionization. However, we have found that other fluorinated alcohols can also be utilized for mobile phase preparation and the choice of optimal fluorinated alcohol is determined by the ion-pairing (IP) agent. Although HFIP is a very good choice to be used alongside less hydrophobic IP agents, other fluorinated alcohols such as 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFMIP) can significantly outperform HFIP when used with more hydrophobic IP agents. We also found that more acidic fluorinated alcohols assist with the transfer of oligonucleotides with secondary structure (e.g., folded strands and hairpins) into the gas phase. Graphical Abstract ᅟ.

Chemical biology tools for regulating RAS signaling complexity in space and time.

Rat sarcoma (RAS) family members are small GTPases that control a number of signaling pathways important for normal cellular proliferation. Therefore, it is no surprise that a significant portion of human tumors express constitutively active mutated RAS proteins, which leads to deregulation of RAS signaling pathways, resulting in pathological perturbations of cell growth and death. Although the molecular details of RAS signaling cascades are well understood, there is still a largely unmet need for small molecule probes to control RAS signaling in space and time. More broadly, given the prevalence of mutated RAS in cancer, the need to translate the insights obtained from using small molecule probes into clinically useful drugs is also significant. In this review, we introduce RAS proteins and the signaling pathways they are involved in, and discuss some of the innovative chemical biology approaches to regulate RAS signaling, which include the exploitation of newly identified binding pockets, covalent inhibitors for mutated RAS, and RAS localization impairment.

Tuning the preference of thiodigalactoside- and lactosamine-based ligands to galectin-3 over galectin-1.

Inhibitors for galectin-1 and -3 were synthesized from thiodigalactoside and lactosamine by derivatization of the galactose C3. Introduction of 4-phenyl-1H-1,2,3-triazol-1-yl substituents at the thiodigalactoside C3 by CuAAC, targeting arginine-arene interactions, increased the affinity to 13 nM but yielded little selectivity. The bulkier 4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl substituent, however, increased the preference for galectin-3 over galectin-1 to more than 200-fold. Modeling showed more arginine-arene interactions for galectin-3 than for galectin-1. Introducing 4-phenoxyaryl groups on lactosamine had a similar effect.