Chemical Biology Approach Research Articles

Enzymatic or In Vivo Installation of Propargyl Groups in Combination with Click Chemistry for the Enrichment and Detection of Methyltransferase Target Sites in RNA.

m6 A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3-METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise transcriptome-wide assignment of m6 A sites is of utmost importance. However, m6 A does not interfere with Watson-Crick base pairing, making polymerase-based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl groups can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65 % at m6 A sites after bioconjugation and purification, hence enabling detection of METTL3-METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl-l-selenohomocysteine and validated different types of known rRNA methylation sites.

High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer

Human Tousled-like kinases (TLKs) are highly conserved serine/threonine protein kinases responsible for cell proliferation, DNA repair, and genome surveillance. Their possible involvement in cancer via efficient DNA repair mechanisms have made them clinically relevant molecular targets for anticancer therapy. Innovative approaches in chemical biology have played a key role in validating the importance of kinases as molecular targets. However, the detailed understanding of the protein structure and the mechanisms of protein–drug interaction through biochemical and biophysical techniques demands a method for the production of an active protein of exceptional stability and purity on a large scale. We have designed a bacterial expression system to express and purify biologically active, wild-type Human Tousled-like Kinase 1B (hTLK1B) by co-expression with the protein phosphatase from bacteriophage λ. We have obtained remarkably high amounts of the soluble and homogeneously dephosphorylated form of biologically active hTLK1B with our unique, custom-built vector design strategy. The recombinant hTLK1B can be used for the structural studies and may further facilitate the development of new TLK inhibitors for anti-cancer therapy using a structure-based drug design approach.

Covalent Allosteric Probe for the Metabotropic Glutamate Receptor2: Design, Synthesis, and Pharmacological Characterization.

Covalent labeling of G protein-coupled receptors (GPCRs) by small molecules is a powerful approach to understand binding modes, mechanism of action, pharmacology, and even facilitate structure elucidation. We report the first covalent positive allosteric modulator (PAM) for a class C GPCR, the mGlu2 receptor. Three putatively covalent mGlu2 PAMs were designed and synthesized. Pharmacological characterization identified 2 to bind the receptor covalently. Computational modeling combined with receptor mutagenesis revealed T7917.29×30 as the likely position of covalent interaction. We show how this covalent ligand can be used to characterize the PAM binding mode and that it is a valuable tool compound in studying receptor function and binding kinetics. Our findings advance the understanding of the mGlu2 PAM interaction and suggest that 2 is a valuable probe for further structural and chemical biology approaches.

Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells

Modular AB-type bacterial protein toxins target mammalian host cells with high specificity and deliver their toxic cargo into the cytosol. Hence, these toxins are being explored as agents for targeted cytosolic delivery in biomedical and research applications. The cytotoxic necrotizing factor (CNF) family is unique among these toxins in that their homologous sequences are found in a wide array of bacteria, and their activity domains are packaged in various delivery systems. Here, to study how CNF cargo and delivery modules can be assembled for efficient cytosolic delivery, we generated chimeric toxins by swapping functional domains among CNF1, CNF2, CNF3, and CNFy. Chimeras with a CNFy delivery vehicle were more stably expressed, but were less efficient at cargo delivery into HEK293-T cells. We also found that CNFy cargo is the most universally compatible and that CNF3 delivery vehicle is the most flexible and efficient at delivering cargo. These findings suggest that domains within proteins can be swapped and accommodate each other for efficient function and that an individual domain could be engineered for compatibility with multiple partner domains. We anticipate that our insights could help inform chemical biology approaches to develop toxin-based cargo-delivery platforms for cytosolic cargo delivery of therapeutics or molecular probes into mammalian cells.

Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology.

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control in situ the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H3 receptor (H3R). From 16 newly synthesized photoswitchable compounds, VUF14738 (28) and VUF14862 (33) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H3R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical trans-/cis conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H3R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H3R signaling.

Cystathionine β-Synthase Is Necessary for Axis Development in Vivo.

The cystathionine ß-synthase (CBS) is a critical enzyme in the transsulfuration pathway and is responsible for the synthesis of cystathionine from serine and homocysteine. Cystathionine is a precursor to amino acid cysteine. CBS is also responsible for generation of hydrogen sulfide (H2S) from cysteine. Mutation in CBS enzyme causes homocysteine levels to rise, and gives rise to a condition called hyperhomocysteinuria. To date, numerous mouse knockout models for CBS enzyme has been generated, which show panoply of defects, reflecting the importance of this enzyme in development. In zebrafish, we and others have identified two orthologs of cbs, which we call cbsa and cbsb. Previous gene knockdown studies in zebrafish have reported a function for cbsb ortholog in maintaining ion homeostasis in developing embryos. However, its role in maintaining H2S homeostasis in embryos is unknown. Here, we have performed RNA analysis in whole zebrafish embryos that showed a wide expression pattern for cbsa and cbsb primarily along the embryonic axis of the developing embryo. Loss-of-function analysis using a combination of approaches which include splice morpholinos and CRISPR/Cas9 genomic engineering show evidence that cbsb ortholog is responsible for anterior-posterior axis development, and cbsa function is redundant. Cbsb loss of function fish embryos show shortened and bent axis, along with less H2S and more homocysteine, effects resulting from loss of Cbsb. Using a chemical biology approach, we rescued the axis defects with betaine, a compound known to reduce homocysteine levels in plasma, and GYY4137, a long term H2S donor. These results collectively argue that cells along the axis of a developing embryo are sensitive to changes in homocysteine and H2S levels, pathways that are controlled by Cbsb, and thus is essential for development.

Identifizierung von Substratbindestellen in der γ-Sekretase

The lack of detailed information on the interactions between substrates and enzymes is a major gap in understanding the mechanisms of many proteases. Using the Alzheimer’s disease protease γ-secretase as an example, we show how a previously developed versatile chemical biology approach allows to systematically identify molecular interactions with the enzyme at the amino acid level of the substrate. Furthermore, this approach provides additional mechanistic insights that could also aid in the development of advanced drugs against this key protease.

Opportunities for Lipid-Based Probes in the Field of Immunology.

Lipids perform a wide range of functions inside the cell, ranging from structural building block of membranes and energy storage to cell signaling. The mode of action of many signaling lipids has remained elusive due to their low abundance, high lipophilicity, and inherent instability. Various chemical biology approaches, such as photoaffinity or activity-based protein profiling methods, have been employed to shed light on the biological role of lipids and the lipid-protein interaction profile. In this review, we will summarize the recent developments in the field of chemical probes to study lipid biology, especially in immunology, and indicate potential avenues for future research.

Abstract LB-B28: Abietane natural products as novel therapeutics against solid tumors

Abstract Dysregulation of protein synthesis can lead to aberrant cellular proliferation and survival, a hallmark of cancer. Protein synthesis is a multistep process tightly controlled by specific translation factors. In eukaryotes, at the initiation point, the 43S preinitiation complex is formed by the association of several factors [eukaryotic initiation factor 2 (eIF2), GTP and methionyl initiator tRNA] with the 40S ribosomal subunit and the eukaryotic initiation factor 3 (eIF3). Over expression of eIFs has been reported in breast cancer, particularly in the most aggressive subtype, triple negative breast cancer (TNBC). More than one million global cases of breast cancer are diagnosed yearly and approximately 15% are characterized as triple negative breast cancer (TNBC), a subtype that lacks effective targeted therapy modalities. Recently, we generated a library of cell permeable abietane natural product derivatives (i.e. SJ6038) and showed that these compounds are more potent against TNBC over the estrogen receptor positive cellular models. These compounds were tagged with biotin for affinity enrichment and with a fluorescent chromophore to facilitate protein target(s) identification. A SILAC proteomics approach was used to distinguish specific binding targets from the non-specific ones. Gene Ontology analysis showed the targets identified were affiliated with regulation of protein synthesis. Ingenuity Pathway Analysis suggested that abietanes may exert their anticancer effects on critical biological pathways such as the eIF2, eIF4/p70S6K, and mTOR signaling pathways. Live cell experiments utilizing fluorescent abietane derivatives demonstrate that these probes localize to subsections of the ER, supporting the SILAC findings. Time of Exposure and Dose Response experiments further demonstrate that Abietane derivatives affect the eIF2 signaling axis. Using chemical biology and medicinal chemistry approaches, we show that abietane natural products preferentially promote cell death in TNBC cellular models by targeting the eIF2 signaling axis. These compounds will serve as unique chemical tools targeting the eIF2/eIF4 signaling axis and advance from hit- to lead for further preclinical development as a targeted therapy against aggressive solid tumors. Citation Format: Walter Lang, Taotao Ling, Jangsoon Lee, Naoto T. Ueno, Fatima Rivas. Abietane natural products as novel therapeutics against solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B28.

Isoform-selective activity-based profiling of ERK signaling.

Extracellular signal-regulated kinases (ERKs) mediate downstream signaling of RAS-RAF-MEK as key regulators of the mitogen-activated protein kinase (MAPK) pathway. Activation of ERK signaling is a hallmark of cancer and upstream MAPK proteins have been extensively pursued as drug targets for cancer therapies. However, the rapid rise of resistance to clinical RAF and MEK inhibitors has prompted interest in targeting ERK (ERK1 and ERK2 isoforms) directly for cancer therapy. Current methods for evaluating activity of inhibitors against ERK isoforms are based primarily on analysis of recombinant proteins. Strategies to directly and independently profile native ERK1 and ERK2 activity would greatly complement current cell biological tools used to probe and target ERK function. Here, we present a quantitative chemoproteomic strategy that utilizes active-site directed probes to directly quantify native ERK activity in an isoform-specific fashion. We exploit a single isoleucine/leucine difference in ERK substrate binding sites to enable activity-based profiling of ERK1 versus ERK2 across a variety of cell types, tissues, and species. We used our chemoproteomic strategy to determine potency and selectivity of academic (VX-11e) and clinical (Ulixertinib) ERK inhibitors. Correlation of potency estimates by chemoproteomics with anti-proliferative activity of VX-11e and Ulixertinib revealed that >90% inactivation of both native ERK1 and ERK2 is needed to mediate cellular activity of inhibitors. Our findings introduce one of the first assays capable of independent evaluation of native ERK1 and ERK2 activity to advance drug discovery of oncogenic MAPK pathways.

ECOTOXICITY AND GENOTOXICITY OF STEEL SLAGS: PRELIMINARY RESULTS

The European iron and steel industry produces a considerable amount of waste and by-products. Also in Italy the steel slag production is very high. Steel slags may be reused in recycled materials, such as materials for the construction industry, road base and asphalt mixtures, allowing to reduce the final disposal in a landfill. The reuse of this recycled material may generate potential release of toxic compounds for the environment and humans. The aim of this study was to assess the ecotoxicity and genotoxicity of steel slags by using an integrated chemical-biological approach. Chemical analysis of leachates obtained by using short-term leaching tests (UNI EN 12457-2) were performed, to evaluate the waste potential reuse according to the Italian legislation (Ministerial Decree 186/2006). Moreover, solutions obtained from leaching tests were assayed by using ecotoxicity tests on plant and animal organisms and genotoxicity tests on bacteria, plant and human cells. Chemical analyses of the eluates were within the limits of the Italian legislation. The preliminary results of the ecotoxicity and the genotoxicity tests demonstrated that this material has a low toxicity and therefore its potential use as a recycled material.

Chemical Biology Approaches for Investigating the Functions of Lysine Acetyltransferases.

The side-chain acetylation of lysine residues in histones and non-histone proteins catalyzed by lysine acetyltransferases (KATs) represents a widespread posttranslational modification (PTM) in the eukaryotic cells. Lysine acetylation plays regulatory roles in major cellular pathways inside and outside the nucleus. In particular, KAT-mediated histone acetylation has an effect on all DNA-templated epigenetic processes. Aberrant expression and activation of KATs are commonly observed in human diseases, especially cancer. In recent years, the study of KAT functions in biology and disease has greatly benefited from chemical biology tools and strategies. In this Review, we present the past and current accomplishments in the design of chemical biology approaches for the interrogation of KAT activity and function. These methods and probes are classified according to their mechanisms of action and respective applications, with both strengths and limitations discussed.

O-GalNAcylation of RANTES Improves Its Properties as a Human Immunodeficiency Virus Type 1 Entry Inhibitor.

Many human proteins have the potential to be developed as therapeutic agents. However, side effects caused by direct administration of natural proteins have significantly slowed expansion of protein therapeutics into the clinic. Post-translational modifications (PTMs) can improve protein properties, but because of significant knowledge gaps, we are considerably limited in our ability to apply PTMs to generate better protein therapeutics. Here, we seek to fill the gaps by studying the PTMs of a small representative chemotactic cytokine, RANTES. RANTES can inhibit HIV-1 infection by competing with it for binding to receptor CCR5 and stimulating CCR5 endocytosis. Unfortunately, RANTES can induce strong signaling, leading to severe inflammatory side effects. We apply a chemical biology approach to explore the potential of post-translationally modified RANTES as safe inhibitors of HIV-1 infection. We synthesized and systematically tested a library of RANTES isoforms for their ability to inhibit inflammatory signaling and prevent HIV-1 infection of primary human cells. Through this research, we revealed that most of the glycosylated variants have decreased inflammation-associated properties and identified one particular glyco variant, a truncated RANTES containing a Galβ1-3GalNAc disaccharide α-linked to Ser4, which stands out as having the best overall properties: relatively high HIV-1 inhibition potency but also weak inflammatory properties. Moreover, our results provided a structural basis for the observed changes in the properties of RANTES. Taken together, this work highlights the potential importance of glycosylation as an alternative strategy for developing CCR5 inhibitors to treat HIV-1 infection and, more generally, for reducing or eliminating unwanted properties of therapeutic proteins.

Advances in the Chemical Biology of Desferrioxamine B.

Desferrioxamine B (DFOB) was discovered in the late 1950s as a hydroxamic acid metabolite of the soil bacterium Streptomyces pilosus. The exquisite affinity of DFOB for Fe(III) identified its potential for removing excess iron from patients with transfusion-dependent hemoglobin disorders. Many studies have used semisynthetic chemistry to produce DFOB adducts with new properties and broad-ranging functions. More recent approaches in chemical biology have revealed some nuances of DFOB biosynthesis and discovered new DFOB-derived drugs and radiometal imaging agents. The current and potential applications of DFOB continue to inspire a rich body of chemical biology research focused on this bacterial metabolite.

Regulation of glycosaminoglycan biogenesis is critical for sensitive-period-dependent vocal ontogeny.

In the brain, the extracellular matrix (ECM) plays a central role during neural development and thus modulates critical-period regulated behavioral ontogeny. The major components of the ECM are glycosaminoglycans (GAGs) including chondroitin sulfate (CS). However, the specific roles of GAGs in behavioral development are largely unknown. It has been shown that xylosides affect the biological functions of GAGs through modulating GAG biosynthesis. Particularly, xylosides affect GAG biosynthesis through priming of GAG chains (priming activity), competing with endogenous core proteins that carry GAG initiation sites (decoy activity), or both. Using birdsong as our model, we investigated, for the first time, how xyloside-mediated modulation of GAG biogenesis affects song development. Xylosides infused into motor cortex of juvenile birds alter song development by specifically affecting ontogeny of the stereotyped sequence rather than the acoustic structure of syllables. Further analyses reveal that observed changes can be attributed to the priming activity rather than the decoy activity of xylosides. Collectively, these results suggest that regulation of GAG biogenesis through chemical biology approaches may allow promising therapeutic interventions of critical-period-dependent central nervous system plasticity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1401-1412, 2017.

Phenazine carboxylic acid and its derivative induce osteoblast differentiation in preosteoblastic MC3T3-E1 cells but adipocyte differentiation in pluripotent mesenchymal C3H10T1/2 cells.

Osteoblast and adipocyte are differentiated from mesenchymal stem cells and dysregulation of the differentiation might result in disease, such as osteoporosis and diabetes. To find small compounds that induce osteoblast differentiation, we screened an in-house natural compounds library with mouse preosteoblastic MC3T3-E1 cells using alkaline phosphatase (ALP) expression as an early osteoblast marker. We found that phenazine-1-carboxylic acid (PCA), one of the major phenazine derivatives produced by Pseudomonas, induced osteoblast differentiation in the cells at micromolar concentrations. PCA acted synergistically with an agonist of hedgehog signaling in inducing ALP activity in the cells. We also found that 2-hydroxy-PCA (2H-PCA) induced osteoblast differentiation in the cells but 2-methoxy-PCA and 1-hydroxy-phenazine did not. Unexpectedly, treatment of mouse pluripotent mesenchymal C3H10T1/2 cells with PCA or 2H-PCA induced an obvious morphological change. Oil Red O staining and real-time reverse-transcription PCR analysis revealed that PCA induced not osteoblast differentiation but adipocyte differentiation in C3H10T1/2 cells. These compounds could allow us to investigate the mechanism of osteoblast and adipocyte differentiation in the two model cell systems through a chemical biology approach.

Small molecule alteration of RNA sequence in cells and animals

RNA regulation and maintenance are critical for proper cell function. Small molecules that specifically alter RNA sequence would be exceptionally useful as probes of RNA structure and function or as potential therapeutics. Here, we demonstrate a photochemical approach for altering the trinucleotide expanded repeat causative of myotonic muscular dystrophy type 1 (DM1), r(CUG)exp. The small molecule, 2H-4-Ru, binds to r(CUG)exp and converts guanosine residues to 8-oxo-7,8-dihydroguanosine upon photochemical irradiation. We demonstrate targeted modification upon irradiation in cell culture and in Drosophila larvae provided a diet containing 2H-4-Ru. Our results highlight a general chemical biology approach for altering RNA sequence in vivo by using small molecules and photochemistry. Furthermore, these studies show that addition of 8-oxo-G lesions into RNA 3′ untranslated regions does not affect its steady state levels.

Methyl 3-(3-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)-propanamido)benzoate as a Novel and Dual Malate Dehydrogenase (MDH) 1/2 Inhibitor Targeting Cancer Metabolism.

Previously, we reported a hypoxia-inducible factor (HIF)-1 inhibitor LW6 containing an (aryloxyacetylamino)benzoic acid moiety inhibits malate dehydrogenase 2 (MDH2) using a chemical biology approach. Structure-activity relationship studies on a series of (aryloxyacetylamino)benzoic acids identified selective MDH1, MDH2, and dual inhibitors, which were used to study the relationship between MDH enzyme activity and HIF-1 inhibition. We hypothesized that dual inhibition of MDH1 and MDH2 might be a powerful approach to target cancer metabolism and selected methyl-3-(3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamido)-benzoate (16c) as the most potent dual inhibitor. Kinetic studies revealed that compound 16c competitively inhibited MDH1 and MDH2. Compound 16c inhibited mitochondrial respiration and hypoxia-induced HIF-1α accumulation. In xenograft assays using HCT116 cells, compound 16c demonstrated significant in vivo antitumor efficacy. This finding provides concrete evidence that inhibition of both MDH1 and MDH2 may provide a valuable platform for developing novel therapeutics that target cancer metabolism and tumor growth.

Mass spectrometry for fragment screening.

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening.

Integrative approaches in HIV‐1 non‐nucleoside reverse transcriptase inhibitor design

The design of inhibitors for human immunodeficiency virus type‐1 reverse transcriptase (HIV‐1 RT) is one of the most successful approaches for the treatment of HIV infections. Among the HIV‐1 RT inhibitors, non‐nucleoside reverse transcriptase inhibitors (NNRTIs) constitute a prominent drug class, which includes nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine approved for clinical use. However, the efficiency of many of these drugs has been undermined by drug‐resistant variants of HIV‐1 RT, and it therefore becomes inevitable to design novel drugs to cope with resistance. Here, we discuss various drug design strategies, which include traditional medicinal chemistry, computational chemistry, and chemical biology approaches. In particular, computational modeling approaches, including machine learning, empirical descriptors‐based, force‐field, ab initio, and hybrid quantum mechanics/molecular mechanics‐based methods are discussed in detail. We foresee that these methods will have a major impact on efforts to guide the design and discovery of the next generation of NNRTIs that combat RT multidrug resistance. WIREs Comput Mol Sci 2018, 8:e1328. doi: 10.1002/wcms.1328This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics Computer and Information Science > Chemoinformatics Molecular and Statistical Mechanics > Free Energy Methods