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Cell Chemical Biology

Martin Kurnik, Cagla Sahin, Camilla Bertel Andersen, Nikolai Lorenzen, Lise Giehm, Hossein Mohammad-Beigi, Christian Moestrup Jessen, Jan Skov Pedersen, Gunna Christiansen, Steen Vang Petersen, Roland Staal, Girija Krishnamurthy, Keith Pitts, Peter H Reinhart, Frans A A Mulder, Scot Mente, Warren D Hirst, Daniel E Otzen
α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity...
August 29, 2018: Cell Chemical Biology
Max Berman Ferretti, Jennifer Louise Barre, Katrin Karbstein
Consistent with its location on the ribosome, reporter assays demonstrate a role for Rps26 in recognition of the Kozak sequence. Consequently, Rps26-deficient ribosomes display preference for mRNAs encoding components of the high salt and high pH stress response pathways and accumulate in yeast exposed to high salt or pH. Here we use this information to reprogram the cellular response to high salt by introducing point mutations in the Kozak sequence of key regulators for the cell wall MAP-kinase, filamentation, or DNA repair pathways...
August 24, 2018: Cell Chemical Biology
Christine A Marian, Mateusz Stoszko, Lili Wang, Matthew W Leighty, Elisa de Crignis, Chad A Maschinot, Jovylyn Gatchalian, Benjamin C Carter, Basudev Chowdhury, Diana C Hargreaves, Jeremy R Duvall, Gerald R Crabtree, Tokameh Mahmoudi, Emily C Dykhuizen
The persistence of a pool of latently HIV-1-infected cells despite combination anti-retroviral therapy treatment is the major roadblock for a cure. The BAF (mammalian SWI/SNF) chromatin remodeling complex is involved in establishing and maintaining viral latency, making it an attractive drug target for HIV-1 latency reversal. Here we report a high-throughput screen for inhibitors of BAF-mediated transcription in cells and the subsequent identification of a 12-membered macrolactam. This compound binds ARID1A-specific BAF complexes, prevents nucleosomal positioning, and relieves transcriptional repression of HIV-1...
August 23, 2018: Cell Chemical Biology
Arne Fabritius, David Ng, Andreas Michael Kist, Mutlu Erdogan, Ruben Portugues, Oliver Griesbeck
Protein engineering involves generating and screening large numbers of variants for desired properties. While modern DNA technology has made it easy to create protein diversity on the DNA level, the selection and validation of candidate proteins from large libraries remains a challenge. We built a screening platform that integrates high-quality fluorescence-based image analysis and robotic picking of bacterial colonies. It allows tracking each individual colony in a large population and collecting quantitative information on library composition during the protein evolution process...
August 22, 2018: Cell Chemical Biology
Shahzada Khan, Shigemoto Fujii, Tetsuro Matsunaga, Akira Nishimura, Katsuhiko Ono, Tomoaki Ida, Khandaker Ahtesham Ahmed, Tatsuya Okamoto, Hiroyasu Tsutsuki, Tomohiro Sawa, Takaaki Akaike
Reactive persulfides such as cysteine persulfide and glutathione persulfide are produced by bacteria including Salmonella during sulfur metabolism. The biological significance of bacterial reactive persulfides in host-pathogen interactions still warrants investigation. We found that reactive persulfides produced by Salmonella Typhimurium LT2 regulate macrophage autophagy via metabolizing 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), an electrophilic product of reactive oxygen species and nitric oxide signaling...
August 22, 2018: Cell Chemical Biology
Lin Guo, Wu Yang, Qiang Huang, Jiali Qiang, Jonathan Ross Hart, Wenyuan Wang, Junhao Hu, Jidong Zhu, Nan Liu, Yaoyang Zhang
Selenoproteins, defined by the presence of selenocysteines (Sec), play important roles in a wide range of biological processes. All known selenoproteins are marked by the presence of Sec insertion sequence (SECIS) at their mRNA. The lack of an effective analytical method has hindered our ability to explore the selenoproteome and new selenoproteins beyond SECIS. Here, we develop a Sec-specific mass spectrometry-based technique, termed "SecMS," which allows the systematic profiling of selenoproteomes by selective alkylation of Sec...
August 22, 2018: Cell Chemical Biology
Jing Li, Yaru Zhang, Bruno Da Silva Sil Dos Santos, Feng Wang, Yuyong Ma, Christian Perez, Yanling Yang, Junmin Peng, Seth M Cohen, Tsui-Fen Chou, Stephen T Hilton, Raymond J Deshaies
The 26S proteasome is the major proteolytic machine for breaking down cytosolic and nuclear proteins in eukaryotes. Due to the lack of a suitable assay, it is difficult to measure routinely and quantitatively the breakdown of proteins by the 26S proteasome in vitro. In the present study, we developed an assay to monitor proteasome-mediated protein degradation. Using this assay, we discovered that epidithiodiketopiperazine (ETPs) blocked the degradation of our model substrate in vitro. Further characterization revealed that ETPs inhibited proteasome function by targeting the essential proteasomal deubiquitinase Rpn11 (POH1/PSMD14)...
August 22, 2018: Cell Chemical Biology
Yuki Takamatsu, Debananda Das, Satoru Kohgo, Hironori Hayashi, Nicole S Delino, Stefan G Sarafianos, Hiroaki Mitsuya, Kenji Maeda
4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA/MK-8591), a nucleoside reverse transcriptase inhibitor (NRTI) under clinical trials, is a potent and promising long-acting anti-HIV type 1 (HIV-1) agent. EFdA and its derivatives possess a modified 4'-moiety and potently inhibit the replication of a wide spectrum of HIV-1 strains resistant to existing NRTIs. Here, we report that EFdA and NRTIs with a 4'-ethynyl- or 4'-cyano-moiety exerted activity against HIV-1 with an M184V mutation and multiple NRTI-resistant HIV-1s, whereas NRTIs with other moieties (e...
August 17, 2018: Cell Chemical Biology
Teresa Kaserer, Julian Blagg
The emergence of mutations that confer resistance to molecularly targeted therapeutics is dependent upon the effect of each mutation on drug affinity for the target protein, the clonal fitness of cells harboring the mutation, and the probability that each variant can be generated by DNA codon base mutation. We present a computational workflow that combines these three factors to identify mutations likely to arise upon drug treatment in a particular tumor type. The Osprey-based workflow is validated using a comprehensive dataset of ERK2 mutations and is applied to small-molecule drugs and/or therapeutic antibodies targeting KIT, EGFR, Abl, and ALK...
August 17, 2018: Cell Chemical Biology
Zhenhao Fang, Christopher B Marshall, Tadateru Nishikawa, Alvar D Gossert, Johanna M Jansen, Wolfgang Jahnke, Mitsuhiko Ikura
KRAS is frequently mutated in several of the most lethal types of cancer; however, the KRAS protein has proven a challenging drug target. K-RAS4B must be localized to the plasma membrane by prenylation to activate oncogenic signaling, thus we endeavored to target the protein-membrane interface with small-molecule compounds. While all reported lead compounds have low affinity for KRAS in solution, the potency of Cmpd2 was strongly enhanced when prenylated K-RAS4B is associated with a lipid bilayer. We have elucidated a unique mechanism of action of Cmpd2, which simultaneously engages a shallow pocket on KRAS and associates with the lipid bilayer, thereby stabilizing KRAS in an orientation in which the membrane occludes its effector-binding site, reducing RAF binding and impairing activation of RAF...
August 14, 2018: Cell Chemical Biology
Kristian Mark Jacobsen, Nikolaj Lilholm Villadsen, Thomas Tørring, Camilla Bak Nielsen, Trine Salomón, Morten Muhlig Nielsen, Michail Tsakos, Christian Sibbersen, Carsten Scavenius, Rikke Nielsen, Erik Ilsø Christensen, Paula Fernandez Guerra, Peter Bross, Jakob Skou Pedersen, Jan Johannes Enghild, Mogens Johannsen, Jørgen Frøkiær, Jens Overgaard, Michael R Horsman, Morten Busk, Thomas B Poulsen
The natural product family of macrocyclic lipodepsipeptides containing the 4-amido-2,4-pentadienoate functionality possesses intriguing cytotoxic selectivity toward hypoxic cancer cells. These subpopulations of cancer cells display increased metastatic potential and resistance to chemo- and radiotherapy. In this paper, we present studies on the mechanism of action of these natural products in hypoxic cancer cells and show that this involves rapid and hypoxia-selective collapse of mitochondrial integrity and function...
August 9, 2018: Cell Chemical Biology
Ji Young Hyun, Sanggil Kim, Hyun Soo Lee, Injae Shin
In this study we developed an efficient method to prepare glycoengineered β-N-acetylhexosaminidase containing multiple mannose-6-phosphates (M6Ps) by combining genetic code expansion with bioorthogonal ligation techniques. We found that multiple M6P-conjugated enzymes were produced with a high efficiency by using combined techniques. Importantly, glycoengineered enzymes entered lysosomes of patient-derived primary cells, which lack endogenous lysosomal β-N-acetylhexosaminidase, more readily than commercialized human β-hexosaminidase...
August 8, 2018: Cell Chemical Biology
Francesco Balestri, Luca Quattrini, Vito Coviello, Stefania Sartini, Federico Da Settimo, Mario Cappiello, Roberta Moschini, Antonella Del Corso, Umberto Mura, Concettina La Motta
Aldose reductase (AKR1B1), the key enzyme of the polyol pathway, plays a crucial role in the development of long-term complications affecting diabetic patients. Nevertheless, the expedience of inhibiting this enzyme to treat diabetic complications has failed, due to the emergence of side effects from compounds under development. Actually AKR1B1 is a Janus-faced enzyme which, besides ruling the polyol pathway, takes part in the antioxidant defense mechanism of the body. In this work we report the evidence that a class of compounds, characterized by a pyrazolo[1,5-a]pyrimidine core and an ionizable fragment, modulates differently the catalytic activity of the enzyme, depending on the presence of specific substrates such as sugar, toxic aldehydes, and glutathione conjugates of toxic aldehydes...
August 4, 2018: Cell Chemical Biology
Giovanna Scapin, Clinton S Potter, Bridget Carragher
We present a perspective of our view of the application of cryoelectron microscopy (cryo-EM) to structure-based drug design (SBDD). We discuss the basic needs and requirements for SBDD, the current state of cryo-EM, and the challenges that need to be overcome for this technique to reach its full potential in facilitating the process of drug discovery.
July 26, 2018: Cell Chemical Biology
Zhenyi Hu, Hiromi Tanji, Shuangshuang Jiang, Shuting Zhang, Kyoin Koo, Jean Chan, Kentaro Sakaniwa, Umeharu Ohto, Albert Candia, Toshiyuki Shimizu, Hang Yin
Rational design of drug-like small-molecule ligands based on structural information of proteins remains a significant challenge in chemical biology. In particular, designs targeting protein-protein interfaces have met little success given the dynamic nature of the protein surfaces. Herein, we utilized the structure of a small-molecule ligand in complex with Toll-like receptor 8 (TLR8) as a model system due to TLR8's clinical relevance. Overactivation of TLR8 has been suggested to play a prominent role in the pathogenesis of various autoimmune diseases; however, there are still few small-molecule antagonists available, and our rational designs led to the discovery of six exceptionally potent compounds with ∼picomolar IC50 values...
July 25, 2018: Cell Chemical Biology
Zhengliang L Wu, Timothy J Tatge, Alex E Grill, Yonglong Zou
O-GlcNAcylation is a reversible serine/threonine glycosylation for regulating protein activity and availability inside cells. In a given protein, O-GlcNAcylated and unoccupied O-linked β-N-acetylglucosamine (O-GlcNAc) sites are referred to as closed and open sites, respectively. The balance between open and closed sites is believed to be dynamically regulated. In this report, closed sites are detected using in vitro incorporation of GalNAz by B3GALNT2, and open sites are detected by in vitro incorporation of GlcNAz by O-GlcNAc transferase (OGT), via click chemistry...
July 24, 2018: Cell Chemical Biology
Natalia Rinis, Jennifer E Golden, Caleb D Marceau, Jan E Carette, Michael C Van Zandt, Reid Gilmore, Joseph N Contessa
The oligosaccharyltransferase (OST) is a multisubunit enzyme complex that N-glycosylates proteins in the secretory pathway and is considered to be constitutive and unregulated. However, small-molecule OST inhibitors such as NGI-1 provide a pharmacological approach for regulating N-linked glycosylation. Herein we design cell models with knockout of each OST catalytic subunit (STT3A or STT3B) to screen the activity of NGI-1 and its analogs. We show that NGI-1 targets the function of both STT3A and STT3B and use structure-activity relationships to guide synthesis of catalytic subunit-specific inhibitors...
July 24, 2018: Cell Chemical Biology
James S Italia, Christopher Latour, Chester J J Wrobel, Abhishek Chatterjee
The bacteria-derived tyrosyl-tRNA synthetase (TyrRS)/tRNA pair was first used for unnatural amino acid (Uaa) mutagenesis in eukaryotic cells over 15 years ago. It provides an ideal platform to genetically encode numerous useful Uaas in eukaryotes. However, this pair has been engineered to charge only a small collection of Uaas to date. Development of Uaa-selective variants of this pair has been limited by technical challenges associated with a yeast-based directed evolution platform, which is currently required to alter its substrate specificity...
July 18, 2018: Cell Chemical Biology
Chloe J Peach, Laura E Kilpatrick, Rachel Friedman-Ohana, Kris Zimmerman, Matthew B Robers, Keith V Wood, Jeanette Woolard, Stephen J Hill
Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF165 a, VEGF165 b, and VEGF121 a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2...
July 18, 2018: Cell Chemical Biology
Kabirul Islam
Successful mapping of the human genome has sparked a widespread interest in deciphering functional information encoded in gene sequences. However, because of the high degree of conservation in sequences along with topological and biochemical similarities among members of a protein superfamily, uncovering physiological role of a particular protein has been a challenging task. Chemical genetic approaches have made significant contributions toward understanding protein function. One such effort, dubbed the bump-and-hole approach, has convincingly demonstrated that engineering at the protein-small molecule interface constitutes a powerful method for elucidating the function of a specific gene product...
July 16, 2018: Cell Chemical Biology
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