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Ribonuclease targeting chimeras (RiboTACs) induce degradation of an RNA target by facilitating an interaction between an RNA and a ribonuclease (RNase). We describe the screening of a DNA-encoded library (DEL) to identify binders of monomeric RNase L to provide a compound that induced dimerization of RNase L, activating its ribonuclease activity. This compound was incorporated into the design of a next-generation RiboTAC that targeted the microRNA-21 (miR-21) precursor and alleviated a miR-21-associated cellular phenotype in triple-negative breast cancer cells. The RNA-binding module in the RiboTAC is Dovitinib, a known receptor tyrosine kinase (RTK) inhibitor, which was previously identified to bind miR-21 as an off-target. Conversion of Dovitinib into this RiboTAC reprograms the known drug to selectively affect the RNA target. This work demonstrates that DEL can be used to identify compounds that bind and recruit proteins with effector functions in heterobifunctional compounds.

The interactions between cellular RNAs in MDA-MB-231 triple negative breast cancer cells and a panel of small molecules appended with a diazirine cross-linking moiety and an alkyne tag were probed transcriptome-wide in live cells. The alkyne tag allows for facile pull-down of cellular RNAs bound by each small molecule, and the enrichment of each RNA target defines the compound’s molecular footprint. Among the 34 chemically diverse small molecules studied, six bound and enriched cellular RNAs. The most highly enriched interaction occurs between the novel RNA binding compound F1 and a structured region in the 5′ untranslated region of quiescin sulfhydryl oxidase 1 isoform a (QSOX1-a), not present in isoform b. Additional studies show that F1 specifically bound RNA over DNA and protein; that is, we studied the entire DNA, RNA, and protein interactome. This interaction was used to design a ribonuclease targeting chimera (RIBOTAC) to locally recruit Ribonuclease L to degrade QSOX1 mRNA in an isoform-specific manner, as QSOX1-a, but not QSOX1-b, mRNA and protein levels were reduced. The RIBOTAC alleviated QSOX1-mediated phenotypes in cancer cells. This approach can be broadly applied to discover ligands that bind RNA in cells, which could be bioactive themselves or augmented with functionality such as targeted degradation.

The discovery of biofunctional natural products (NPs) has relied on the phenotypic screening of extracts and subsequent laborious work to dereplicate active NPs and define cellular targets. Herein, NPs present as crude extracts, partially purified fractions, and pure compounds were screened directly against molecular target libraries of RNA structural motifs in a library-versus-library fashion. We identified 21 hits with affinity for RNA, including one pure NP, nocathiacin I (NOC-I). The resultant data set of NOC-I-RNA fold interactions was mapped to the human transcriptome to define potential bioactive interactions. Interestingly, one of NOC-I's most preferred RNA folds is present in the nuclease processing site in the oncogenic, noncoding microRNA-18a, which NOC-I binds with low micromolar affinity. This affinity for the RNA translates into the selective inhibition of its nuclease processing in vitro and in prostate cancer cells, in which NOC-I also triggers apoptosis. In principle, adaptation of this combination of experimental and predictive approaches to dereplicate NPs from the other hits (extracts and partially purified fractions) could fundamentally transform the current paradigm and accelerate the discovery of NPs that bind RNA and their simultaneous correlation to biological targets.

Arbidol (ARB) is a broad-spectrum antiviral drug approved in Russia and China for the treatment of influenza. ARB was tested in patients as a drug candidate for the treatment at the early onset of COVID-19 caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite promising clinical results and multiple ongoing trials, preclinical data are lacking and the molecular mechanism of action of ARB against SARS-CoV-2 remains unknown. Here, we demonstrate that ARB binds to the spike viral fusion glycoprotein of the SARS-CoV-2 Wuhan strain as well as its more virulent variants from the United Kingdom (strain B.1.1.7) and South Africa (strain B.1.351). We pinpoint the ARB binding site on the S protein to the S2 membrane fusion domain and use an infection assay with Moloney murine leukemia virus (MLV) pseudoviruses (PVs) pseudotyped with the S proteins of the Wuhan strain and the new variants to show that this interaction is sufficient for the viral cell entry inhibition by ARB. Finally, our experiments reveal that the ARB interaction leads to a significant destabilization and eventual lysosomal degradation of the S protein in cells. Collectively, our results identify ARB as the first clinically approved small molecule drug binder of the SARS-CoV-2 S protein and place ARB among the more promising drug candidates for COVID-19.

Reprogramming known medicines for a novel target with activity and selectivity over the canonical target is challenging. By studying the binding interactions between RNA folds and known small-molecule medicines and mining the resultant dataset across human RNAs, we identified that Dovitinib, a receptor tyrosine kinase (RTK) inhibitor, binds the precursor to microRNA-21 (pre-miR-21). Dovitinib was rationally reprogrammed for pre-miR-21 by using it as an RNA recognition element in a chimeric compound that also recruits RNase L to induce the RNA's catalytic degradation. By enhancing the inherent RNA-targeting activity and decreasing potency against canonical RTK protein targets in cells, the chimera shifted selectivity for pre-miR-21 by 2500-fold, alleviating disease progression in mouse models of triple-negative breast cancer and Alport Syndrome, both caused by miR-21 overexpression. Thus, targeted degradation can dramatically improve selectivity even across different biomolecules, i.e., protein versus RNA.

The curcusone natural products are complex diterpenes featuring a characteristic [6-7-5] tricyclic carbon skeleton similar to the daphnane and tigliane diterpenes. Among them, curcusones A-D demonstrated potent anticancer activity against a broad spectrum of human cancer cell lines. Prior to this study, no total synthesis of the curcusones was achieved and their anticancer mode of action remained unknown. Herein, we report our synthetic and chemoproteomics studies of the curcusone diterpenes that culminate in the first total synthesis of several curcusone natural products and identification of BRCA1-associated ATM activator 1 (BRAT1) as a cellular target. Our efficient synthesis is highly convergent, builds upon cheap and abundant starting materials, features a thermal [3,3]-sigmatropic rearrangement and a novel FeCl(3)-promoted cascade reaction to rapidly construct the critical cycloheptadienone core of the curcusones, and led us to complete the first total synthesis of curcusones A and B in only 9 steps, C and D in 10 steps, and dimericursone A in 12 steps. The chemical synthesis of dimericursone A from curcusones C and D provided direct evidence to support the proposed Diels-Alder dimerization and cheletropic elimination biosynthetic pathway. Using an alkyne-tagged probe molecule, BRAT1, an important but previously “undruggable” oncoprotein, was identified as a key cellular target via chemoproteomics. We further demonstrate for the first time that BRAT1 can be inhibited by curcusone D, resulting in impaired DNA damage response, reduced cancer cell migration, potentiated activity of the DNA damaging drug etoposide, and other phenotypes similar to BRAT1 knockdown.

Herein, we report arylazopyrazole ureas and sulfones as a novel class of photoswitchable serine hydrolase inhibitors and present a chemoproteomic platform for rapid discovery of optically controlled serine hydrolase targets in complex proteomes. Specifically, we identify highly potent and selective photoswitchable inhibitors of the drug-metabolizing enzymes carboxylesterases 1 and 2 and demonstrate their pharmacological application by optically controlling the metabolism of the immunosuppressant drug mycophenolate mofetil. Collectively, this proof-of-concept study provides a first example of photopharmacological tools to optically control drug metabolism by modulating the activity of a metabolizing enzyme. Our arylazopyrazole ureas and sulfones offer synthetically accessible scaffolds that can be expanded to identify specific photoswitchable inhibitors for other serine hydrolases, including lipases, peptidases, and proteases. Our chemoproteomic platform can be applied to other photoswitches and scaffolds to achieve optical control over diverse protein classes.

Many RNAs are processed into biologically active transcripts, the aberrant expression of which can contribute to disease phenotypes. For example, the primary microRNA-17-92 (pri-miR-17-92) cluster contains six microRNAs (miRNAs) that collectively act in several disease settings. Herein, we used sequence-based design of structure-specific ligands to target a common structure in the Dicer processing sites of three miRNAs in the cluster, miR-17, miR-18a, and miR-20a, thereby inhibiting their biogenesis. The compound was optimized to afford a dimeric molecule that binds the Dicer processing site and an adjacent bulge, affording a 100-fold increase in potency. The dimer's mode of action was then extended from simple binding to direct cleavage by conjugation to bleomycin A5 in a manner that imparts RNA-selective cleavage or to indirect cleavage by recruiting an endogenous nuclease, or a ribonuclease targeting chimera (RIBOTAC). Interestingly, the dimer-bleomycin conjugate cleaves the entire pri-miR-17-92 cluster and hence functionally inhibits all six miRNAs emanating from it. The compound selectively reduced levels of the cluster in three disease models: polycystic kidney disease, prostate cancer, and breast cancer, rescuing disease-associated phenotypes in the latter two. Further, the bleomycin conjugate exerted selective effects on the miRNome and proteome in prostate cancer cells. In contrast, the RIBOTAC only depleted levels of pre- and mature miR-17, -18a, and 20a, with no effect on the primary transcript, in accordance with the cocellular localization of RNase L, the pre-miRNA targets, and the compound. These studies demonstrate a strategy to tune RNA structure-targeting compounds to the cellular localization of the target.

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

Serine hydrolases play crucial biological roles and are important therapeutic targets in many clinical applications. Activity-based protein profiling of serine hydrolases by using fluorophosphonate probes, pioneered by Cravatt and co-workers, has been a powerful tool for interrogating serine hydrolases in various biological systems. Herein, we present new phenyl phosphonate probes with an azide handle for click chemistry that offer remarkable improvements over the classical fluorophosphonate serine hydrolase activity-based probes including ease of preparation, excellent cell permeability, and distinct reactivity profiles, as controlled by the phenolate leaving group. Thus, these new activity-based serine hydrolase probes are valuable tools to further interrogate this important class of enzymes.

A small molecule (1) with overlapping affinity for two microRNA (miRNA) precursors was used to inform design of a dimeric compound (2) selective for one of the miRNAs. In particular, 2 selectively targets the microRNA(miR)-515 hairpin precursor to inhibit production of miR-515 that represses sphingosine kinase 1 (SK1), a key enzyme in the biosynthesis of sphingosine 1-phosphate (S1P). Application of 2 to breast cancer cells enhanced SK1 and S1P levels, triggering a migratory phenotype. Knockout of SK1, forced overexpression of miR-515, and application of a small molecule SK1 inhibitor all ablated 2's effect on phenotype, consistent with its designed mode of action. Target profiling studies via Chem-CLIP showed that 2 bound selectively to the miR-515 hairpin precursor in cells. Global neoprotein synthesis upon addition of 2 to MCF-7 breast cancer cells demonstrated 2's selectivity and upregulation of cancer-associated proteins regulated by S1P. The most upregulated protein was human epidermal growth factor receptor 2 (ERBB2/HER2), which is regulated by the SK1/S1P pathway and is normally not expressed in MCF-7 cells. Like triple negative breast cancer (TNBC) cells, the lack of HER2 renders them insusceptible to Herceptin and its antibody-drug conjugate Kadcyla. In addition to proteomics, an RNA-seq study supports that 2 has limited off target effects and other studies support that 2 is more selective than an oligonucleotide. We therefore hypothesized that 2 could sensitize MCF-7 cells to anti-HER2 therapies. Indeed, application of 2 sensitized cells to Herceptin. These results were confirmed in two other cell lines that express miR-515 and are HER2-, the hepatocellular carcinoma cell line HepG2 and the TNBC line MDA-MB-231. Importantly, normal breast epithelial cells (MCF-10A) that do not express miR-515 are not affected by 2. These observations suggest a precision medicine approach to sensitize HER2- cancers to approved anticancer medicines. This study has implications for broadening the therapeutic utility of known targeted cancer therapeutics by using a secondary targeted approach to render otherwise insensitive cells, sensitive to a targeted therapeutic.

The natural product cepafungin I was recently reported to be one of the most potent covalent inhibitors of the 20S proteasome core particle through a series of in vitro activity assays. Here, we report a short chemoenzymatic total synthesis of cepafungin I featuring the use of a regioselective enzymatic oxidation to prepare a key hydroxylated amino acid building block in a scalable fashion. The strategy developed herein enabled access to a chemoproteomic probe, which in turn revealed the exceptional selectivity and potency of cepafungin I towards the b2 and b5 subunits of the proteasome. Further structure-activity relationship studies suggest the key role of the hydroxyl group in the macrocycle and the identity of the lipid tail in modulating the potency of this natural product family. This study lays the groundwork for further medicinal chemistry exploration to fully realize the anticancer potential of cepafungin I.

Significance Parkinson’s disease and its associated dementia can be caused by elevated levels of α-synuclein protein. Despite many efforts, however, targeting α-synuclein at the protein level has been challenging. In this manuscript, we describe the design of a small molecule that selectively targets the messenger RNA that encodes α-synuclein protein and selectively inhibits its translation. Furthermore, the compound is cytoprotective. Collectively, our findings show that difficult-to-target proteins can indeed be regulated by small molecules by binding the encoding mRNA. This strategy is a potentially promising way to slow the progression of Parkinson’s disease and related neurological disorders., Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson’s disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5′ untranslated region (5′ UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5′ UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound’s selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.

Protein S-palmitoylation is a post-translational modification that plays a crucial role in cancer cells by regulating the function and localization of oncoproteins and tumor suppressor proteins. Here, we identify artemisinin (ART), a clinically approved antimalarial endoperoxide natural product with promising anticancer activities, as an inhibitor of the ER-residing palmitoyl transferase ZDHHC6 in cancer cells using a chemoproteomic approach. We show that ART covalently binds and inhibits ZDHHC6 to reduce palmitoylation of the oncogenic protein NRas, disrupt NRas subcellular localization, and attenuate the downstream pro-proliferative signaling cascades. Our study identifies artemisinin as a non-lipid-based palmitoylation inhibitor targeting a specific palmitoyl acyltransferase and provides valuable mechanistic insights into the anticancer activity of artemisinins that are currently being studied in human clinical trials for different cancers.

Summary MicroRNA families are ubiquitous in the human transcriptome, yet targeting of individual members is challenging because of sequence homology. Many secondary structures of the precursors to these miRNAs (pri- and pre-miRNAs), however, are quite different. Here, we demonstrate both in vitro and in cellulis that design of structure-specific small molecules can inhibit a particular miRNA family member to modulate a disease pathway. The miR-200 family consists of five miRNAs, miR-200a, -200b, -200c, -141, and -429, and is associated with type 2 diabetes (T2D). We designed a small molecule that potently and selectively targets pre-miR-200c’s structure and reverses a pro-apoptotic effect in a pancreatic β cell model. In contrast, an oligonucleotide targeting the RNA’s sequence inhibited all family members. Global proteomics and RNA sequencing analyses further demonstrate selectivity for miR-200c. Collectively, these studies establish that miR-200c plays an important role in T2D, and small molecules targeting RNA structure can be an important complement to oligonucleotides.

Abiespiroside A (1), beshanzuenone C (2), and beshanzuenone D (3) belong to the Abies sesquiterpenoid family. Beshanzuenones C (2) and D (3) are isolated from the critically endangered Chinese fir tree species Abies beshanzuensis and demonstrated weak inhibiting activity against protein tyrosine phosphatase 1B (PTP1B). We describe herein the first total syntheses of these Abies sesquiterpenoids relying on the sustainable and inexpensive chiral pool molecule (+)-carvone. The syntheses feature a palladium-catalyzed hydrocarbonylative lactonization to install the 6,6-fused bicyclic ring system and a Dreiding-Schmidt reaction to build the oxaspirolactone moiety of these target molecules. Our chemical total syntheses of these Abies sesquiterpenoids have enabled (i) the validation of beshanzuenone C’s weak PTP1B inhibiting potency, (ii) identification of new synthetic analogs with promising and selective protein tyrosine phosphatase SHP2 inhibiting potency, and (iii) preparation of azide-tagged probe molecules for target identification via a chemoproteomic approach. The latter has resulted in the identification and evaluation of DNA polymerase epsilon subunit 3 (POLE3) as one of the novel cellular targets of these Abies sesquiterpenoids and their analogs. More importantly, via POLE3 inactivation by probe molecule 29 and knockdown experiment, we further demonstrated that targeting POLE3 with small molecules may be a novel strategy for chemosensitization to DNA damaging drugs such as etoposide in cancer.

Changes in cellular sterol species and concentrations can have profound effects on the transcriptional profile. In yeast, mutants defective in sterol biosynthesis show a wide range of changes in transcription, including a coinduction of anaerobic genes and ergosterol biosynthesis genes, biosynthesis of basic amino acids, and several stress genes. However the mechanisms underlying these changes are unknown. We identified mutations in the SAGA complex, a coactivator of transcription, which abrogate the ability to carry out most of these sterol-dependent transcriptional changes. In the erg3 mutant, the SAGA complex increases its occupancy time on many of the induced ergosterol and anaerobic gene promoters, increases its association with several relevant transcription factors and the SWI/SNF chromatin remodeling complex, and surprisingly, associates with an endocytic protein, Rvs167p, suggesting a moonlighting function for this protein in the sterol-regulated induction of the heat shock protein, HSP42 and HSP102, mRNAs.

The disulfide dihedral angle in epidithiodiketopiperazines (ETPs) is near 0°. Application of this highest possible ring tension to strain-promoted thiol-mediated uptake results in efficient delivery to the cytosol and nucleus. Compared to the previous best asparagusic acid (AspA), ring-opening disulfide exchange with ETPs occurs more efficiently even with nonactivated thiols, and the resulting thiols exchange rapidly with nonactivated disulfides. ETP-mediated cellular uptake is more than 20 times more efficient compared to AspA, occurs without endosomal capture, depends on temperature, and is “unstoppable” by inhibitors of endocytosis and conventional thiol-mediated uptake, including siRNA against the transferrin receptor. These results suggest that ETP-mediated uptake not only maximizes delivery to the cytosol and nucleus but also opens the door to a new multitarget hopping mode of action., Epidithiodiketopiperazines are introduced for nontoxic delivery to the cytosol and nucleus, without endosomal capture, unstoppable by conventional inhibitors, opening the doors to an intriguing multitarget disulfide hopping mechanism.

Summary Rpn13 is one of several ubiquitin receptors in the 26S proteasome. Cys88 of Rpn13 has been proposed to be the principal target of RA190, an electrophilic small molecule with interesting anti-cancer activities. Here, we examine the claim that RA190 mediates its cytotoxic effects through engagement with Rpn13. We find no evidence that this is the case. In vitro, RA190 is has no measurable effect on any of the known interactions of Rpn13. In cellulo, we see no physical engagement of Rpn13 by RA190, either on C88 or any other residue. However, chemical proteomics experiments in two different cell lines reveal that dozens of other proteins are heavily engaged by RA190. Finally, increasing or reducing the level of Rpn13 in HeLa and melanoma cells had no effect on the sensitivity of HeLa or melanoma cells to RA190. We conclude that Rpn13 is not the physiologically relevant target of RA190.

Summary In this study, we identify the natural product gambogic acid as well as structurally related synthetic xanthones as first-in-class covalent inhibitors of the de novo sphingolipid biosynthesis. We apply chemoproteomics to determine that gambogic acid binds to the regulatory small subunit B of the serine palmitoyltransferase complex (SPTSSB). We then test structurally related synthetic xanthones to identify 18 as an equally potent but more selective binder of SPTSSB and show that 18 reduces sphingolipid levels in situ and in vivo. Finally, using various biological methods, we demonstrate that 18 induces cellular responses characteristic for diminished sphingosine-1-phosphate (S1P) signaling. This study demonstrates that SPTSSB may become a viable therapeutic target in various diseases with pathological S1P signaling. Furthermore, we believe that our compound will become a valuable tool for studying the sphingolipid metabolism and serve as a blueprint for the development of a new generation of sphingolipid biosynthesis inhibitors.

Proteomic profiling using bioorthogonal chemical probes that selectively react with certain amino acids is now a widely used method in life sciences to investigate enzymatic activities, study posttranslational modifications and discover novel covalent inhibitors. Over the past two decades, researchers have developed selective probes for several different amino acids, including lysine, serine, cysteine, threonine, tyrosine, aspartate and glutamate. Among these amino acids, cysteines are particularly interesting due to their highly diverse and complex biochemical role in our cells. In this feature article, we focus on the chemical probes and methods used to study cysteines in complex proteomes.

In this study, we demonstrate that appendage of a single asparagusic acid residue (AspA tag) is sufficient to ensure efficient cellular uptake and intracellular distribution of fully unprotected peptides. We apply this new delivery method to induce apoptotic response in cancer cells using long (up to 20mer) BH3 domain peptides. Moreover, to understand the molecular mechanism of the cellular uptake, we perform chemical proteomics experiments and identify the direct molecular targets of the asparagusic acid tag. Our findings document covalent bond formation between the asparagusic acid moiety and the cysteines 556 and 558 on the surface of the transferrin receptor resulting in subsequent endocytic uptake of the payload. We believe that the small size, low cellular toxicity and the efficient transferrin receptor-mediated uptake render the AspA tag highly attractive for various life science applications.

Herbal extracts containing sesquiterpene lactones have been extensively used in traditional medicine and are known to be rich in α,β-unsaturated functionalities that can covalently engage target proteins. Here we report synthetic methodologies to access analogues of deoxyelephantopin, a sesquiterpene lactone with anticancer properties. Using alkyne-tagged cellular probes and quantitative proteomics analysis, we identified several cellular targets of deoxyelephantopin. We further demonstrate that deoxyelephantopin antagonizes PPARγ activity in situ via covalent engagement of a cysteine residue in the zinc-finger motif of this nuclear receptor., Deoxyelephantopin is a naturally occurring sesquiterpene lactone with known anticancer properties. Here, the authors synthesize deoxyelephantopins and a range of analogues including alkyne-tagged probes, using them to identify its cellular targets.

The 2016 International Symposium on Chemical Biology was organized by the Swiss National Centre of Competence in Research (NCCR) Chemical Biology. The NCCR Chemical Biology is located at the University of Geneva (UniGE; Director: Prof. Howard Riezman) and the Ecole Polytechnique Fedé rale de Lausanne (EPFL; Co-director: Prof. ́ Kai Johnsson). The symposium was held on January 13−15, 2016 at the Campus Biotech, a new center for biotechnology and life science research that was formed through a unique partnership between the University of Geneva and EPFL and is located within walking distance from the city center of Geneva. This exciting scientific venue attracted more than 240 attendees from around the world. With the aim of providing a sound overview of key challenges in cutting edge chemical biology, as well as fostering an open dialogue with networking opportunities, the organizing committee selected 15 plenary lectures with diverse scope under the broad subject of chemical biology. The scientific program was rounded up by four poster sessions with over 60 posters.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLβ. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here, we report a series of in vivo-active 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLβ in living systems. Optimized inhibitors showed excellent selectivity for DAGLβ over other serine hydrolases, including DAGLα (~60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb−/− mice, we show that DAGLβ inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2 (PLA2G4A). We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLβ is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins and other proteins linked to aberrant cell growth, migration and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.

Serine hydrolases are a diverse enzyme class representing ∼1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,β-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (

SummaryNosocomial infections with the Gram-positive pathogen Clostridium difficile pose a major risk for hospitalized patients and result in significant costs to health care systems. Here, we present the chemical synthesis of a PS-II hapten of a cell wall polysaccharide of hypervirulent ribotype 027 of C. difficile. Mice were immunized with a conjugate consisting of the synthetic hexasaccharide and the diphtheria toxoid variant CRM197. The immunogenicity of the glycan repeating unit was demonstrated by the presence of specific IgG antibodies in the serum of immunized mice. Murine monoclonal antibodies interact with the synthetic hexasaccharide, as determined by microarray analysis. Finally, we found that specific IgA antibodies in the stool of hospital patients infected with C. difficile recognize the synthetic PS-II hexasaccharide hapten.

3-Deoxysugars are important constituents of complex carbohydrates. For example, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) is an essential component of lipopolysaccharides in Gram-negative bacteria, 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN) is widely found in carbohydrates of the bacterial cell wall and in lower vertebrates, and sialic acid is a common cap of mammalian glycoproteins. Although ready access to such sugars would benefit the creation of vaccine candidates, antibiotics and small-molecule drugs, their chemical synthesis is difficult. Here we present a simple chemoenzymatic method for preparing differentially protected 3-deoxysugar derivatives from readily available starting materials. It exploits the promiscuous aldolase activity of the enzyme macrophomate synthase (MPS) to add pyruvate enolate diastereoselectively to a wide range of structurally complex aldehydes. A short synthesis of KDN illustrates the utility of this approach. Enzyme promiscuity, which putatively fosters large functional leaps in natural evolution, has great promise as a source of synthetically useful catalytic transformations.

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.

The process for selecting potent and effective carbohydrate antigens is not well-established. A combination of synthetic glycan microarray screening, surface plasmon resonance analysis, and saturation transfer difference NMR spectroscopy was used to dissect the antibody-binding surface of a carbohydrate antigen, revealing crucial binding elements with atomic-level detail. This analysis takes the first step toward uncovering the rules for structure-based design of carbohydrate antigens.

PEGylated quantum dots (QDs) capped with d-mannose, d-galactose, and d-galactosamine have been synthesized. The stable, high quantum yield fluorescence of QDs was exploited to study specific carbohydrate-protein interactions in vitro and in vivo.

S-palmitoylation involves the attachment of a 16-carbon long fatty acid chain to the cysteine residues of proteins. The process is enzymatic and dynamic with DHHC enzymes mediating palmitoylation and acyl-protein thioesterases reverting the reaction. Proteins that undergo this modification span almost all cellular functions. While the increase in hydrophobicity generated by palmitoylation has the obvious consequence of triggering membrane association, the effects on transmembrane proteins are less intuitive and span a vast range. We review here the current knowledge on palmitoylating and depalmitoylating enzymes, the methods that allow the study of this lipid modification and which drugs can affect it, and finally we focus on four cellular processes for which recent studies reveal an involvement of palmitoylation: endocytosis, reproduction and cell growth, fat and sugar homeostasis and signal transduction at the synapse. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants.

The MPS-catalyzed pyruvate addition is characterized by high diastereoselectivity and mild reaction conditions.

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.

A chemoenzymatic synthesis of sialic acid from inexpensive N-acetyl-D-glucosamine is described. In a three-step Wittig–protection–ozonolysis strategy manno-configured aldehydes are obtained. Treatment with oxaloacetate in the presence of macrophomate synthase affords the signature α-keto-γ-hydroxy acid moiety with high diastereoselectivity.

The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPAL1 and LYPLA2, two enzymes for which selective, in vivo-active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.

A comparative analysis of bacterial and mammalian glycomes based on the statistical analysis of two major carbohydrate databases, Bacterial Carbohydrate Structure Data Base (BCSDB) and GLYCOSCIENCES.de (GS), is presented. An in-depth comparison of these two glycomes reveals both striking differences and unexpected similarities. Within the prokaryotic kingdom, we focus on the glycomes of seven classes of pathogenic bacteria with respect to (i) their most abundant monosaccharide units; (ii) disaccharide pairs; (iii) carbohydrate modifications; (iv) occurrence and use of sialic acids; and (v) class-specific monosaccharides. The aim of this work is to gain insights into unique carbohydrate patterns in bacteria. Data interpretation reveals significant trends in the composition of specific carbohydrate classes as result of evolution-driven structural adaptations of bacterial pathogens and symbionts to their mammalian hosts. The differences are discussed in light of their value for biomedical applications, such as the targeting of unique glycosyl transferases, vaccine development, and devising novel diagnostic tools. peerReviewed

Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.

A short and enantioselective de novo synthesis of an L-glycero-D-manno-heptose building block for the total synthesis of a Yersinia pestis cell wall polysaccharide is described.

A divergent de novo synthesis of six differentially protected l -iduronic acid thioglycosides from a common advanced precursor is described. The key step of this synthetic sequence is the stereoselective elongation of dithioacetal protected C5-dialdehyde 11 via a highly diastereoselective MgBr 2 ·OEt 2 -mediated cyanation. Orthogonally protected l -iduronic acid building blocks obtained by this synthesis are expected to facilitate access to differentially sulfated heparins for microarray-based structure–activity relationship studies.

Vaccinations provide an efficient and cost-effective way to combat devastating human diseases. Besides pathogenic protein markers, cell surface carbohydrates from biological sources are widely used as vaccines. Recently, synthetic immunogenic carbohydrate-protein conjugates have been advanced to vaccine candidates. Progress in the chemical synthesis of oligosaccharides and conjugation methods stimulated the development of novel carbohydrate-based vaccine candidates.

A concise synthesis of a differentially protected d-galacturonic acid (d-GalA) thioglycoside and the construction of a potent immunomodulating glycosphingolipid are described. The key steps of the synthesis are an Evans aldol reaction between a C4 aldehyde and a PMB-protected glycolyloxazolidinone as well as a tandem-PMB-deprotection/cyclization to thioglycosides. The key glycosylation step is optimized by varying the anomeric leaving group, the activating agent, and the solvent system.

The diversity of three major classes of mammalian carbohydrates, mainly glycolipids and O- and N-linked glycans, deposited in the databank GLYCOSCIENCES.de was subjected to statistical analyses. Size, chain length, and branching complexity were accessed and revealed that the average oligosaccharide is composed of about eight monosaccharide units. About a quarter of all oligosaccharides are strictly linear, and the remainder are branched at least once. Glucosamine, galactose, and mannose are dominating and comprise ~75% of the monosaccharides within mammalian oligosaccharide frameworks. alpha-Linked sialic acid, alpha-linked fucose, and beta-linked galactose decorate the majority of reducing termini. Glucose as the most abundant carbohydrate in mammals plays only a very minor role within these structures. Particular emphasis was placed on analyzing the way the monosaccharide units are linked within the oligomeric framework. Just 11 monosaccharide connections account for >75% of all linkages. Thus, the number of structural combinations found in nature, the part of the occupied mammalian glycospace, is much smaller than expected. As a result, a potential set of building blocks for oligosaccharide assembly is presented. This potential building block set was correlated with the accessible 3299 mammalian carbohydrate structures in the GLYCOSCIENCES.de databank. Only 36 building blocks are required to construct 75% of the 3299 mammalian oligosaccharides.