TY  - JOUR
AB  - N-acetyl-l-cysteine (NAC) is a medication and a widely used antioxidant in cell death research. Despite its somewhat obscure mechanism of action, its role in inhibiting ferroptosis is gaining increasing recognition. In this study, we demonstrate that NAC treatment rapidly replenishes the intracellular cysteine pool, reinforcing its function as a prodrug for cysteine. Interestingly, its enantiomer, N-acetyl-d-cysteine (d-NAC), which cannot be converted into cysteine, also exhibits a strong anti-ferroptotic effect. We further clarify that NAC, d-NAC, and cysteine all act as direct reducing substrates for GPX4, counteracting lipid peroxidation. Consequently, only GPX4-rather than system xc-, glutathione biosynthesis, or ferroptosis suppressor protein 1-is necessary for NAC and d-NAC to prevent ferroptosis. Additionally, we identify a broad range of reducing substrates for GPX4 in vitro, including β-mercaptoethanol. These findings provide new insights into the mechanisms underlying the protective effects of NAC and other potential GPX4-reducing substrates against ferroptosis.
AU  - Zheng, J.
AU  - Zhang, W.
AU  - Ito, J.
AU  - Henkelmann, B.
AU  - Xu, C.
AU  - Mishima, E.
AU  - Conrad, M.
C1  - 74302
C2  - 57427
CY  - 50 Hampshire St, Floor 5, Cambridge, Ma 02139 Usa
SP  - 767-775.e5
TI  - N-acetyl-l-cysteine averts ferroptosis by fostering glutathione peroxidase 4.
JO  - Cell Chem. Bio.
VL  - 32
IS  - 5
PB  - Cell Press
PY  - 2025
SN  - 2451-9448
ER  - 

TY  - JOUR
AB  - Respiratory complex I is a multicomponent enzyme conserved between eukaryotic cells and many bacteria, which couples oxidation of electron donors and quinone reduction with proton pumping. Here, we report that protein transport via the Cag type IV secretion system, a major virulence factor of the Gram-negative bacterial pathogen Helicobacter pylori, is efficiently impeded by respiratory inhibition. Mitochondrial complex I inhibitors, including well-established insecticidal compounds, selectively kill H. pylori, while other Gram-negative or Gram-positive bacteria, such as the close relative Campylobacter jejuni or representative gut microbiota species, are not affected. Using a combination of different phenotypic assays, selection of resistance-inducing mutations, and molecular modeling approaches, we demonstrate that the unique composition of the H. pylori complex I quinone-binding pocket is the basis for this hypersensitivity. Comprehensive targeted mutagenesis and compound optimization studies highlight the potential to develop complex I inhibitors as narrow-spectrum antimicrobial agents against this pathogen.
AU  - Lettl, C.*
AU  - Schindele, F.*
AU  - Mehdipour, A.R.*
AU  - Steiner, T.*
AU  - Ring, D.*
AU  - Brack-Werner, R.
AU  - Stecher, B.*
AU  - Eisenreich, W.*
AU  - Bilitewski, U.*
AU  - Hummer, G.*
AU  - Witschel, M.*
AU  - Fischer, W.*
AU  - Haas, R.*
C1  - 67794
C2  - 54272
CY  - 50 Hampshire St, Floor 5, Cambridge, Ma 02139 Usa
SP  - 499-512.e5
TI  - Selective killing of the human gastric pathogen Helicobacter pylori by mitochondrial respiratory complex I inhibitors.
JO  - Cell Chem. Bio.
VL  - 30
IS  - 5
PB  - Cell Press
PY  - 2023
SN  - 2451-9448
ER  - 

TY  - JOUR
AB  - Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.
AU  - Emslander, Q.*
AU  - Vogele, K.*
AU  - Braun, P.*
AU  - Stender, J.*
AU  - Willy, C.*
AU  - Joppich, M.*
AU  - Hammerl, J.A.*
AU  - Abele, M.*
AU  - Meng, C.*
AU  - Pichlmair, A.*
AU  - Ludwig, C.*
AU  - Bugert, J.J.*
AU  - Simmel, F.C.*
AU  - Westmeyer, G.G.
C1  - 65640
C2  - 52845
SP  - 1434-1445.e7
TI  - Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.
JO  - Cell Chem. Bio.
VL  - 29
IS  - 9
PY  - 2022
SN  - 2451-9448
ER  - 

TY  - JOUR
AB  - During insulin resistance, lipid uptake by the liver is promoted by peroxisome proliferator-activated protein (PPAR) γ upregulation, leading to hepatic steatosis. Insulin, however, does not directly regulate adipogenic gene expression in liver, and the mechanisms for its upregulation in obesity remain unclear. Here, we show that the Irs2 locus, a critical regulator of insulin actions, encodes an antisense transcript, ASIrs2, whose expression increases in obesity or after refeeding in liver, reciprocal to that of Irs2. ASIrs2 regulates hepatic Pparg expression, and its suppression ameliorates steatosis in obese mice. The human ortholog AL162497.1, whose expression is correlated with that of hepatic PPARG and the severity of non-alcoholic steatohepatitis (NASH), shows genomic organization similar to that of ASIrs2. We also identified HARS2 as a potential binding protein for ASIrs2, functioning as a regulator of Pparg. Collectively, our data reveal a functional duality of the Irs2 gene locus, where reciprocal changes of Irs2 and ASIrs2 in obesity cause insulin resistance and steatosis.
AU  - Matsushita, M.*
AU  - Awazawa, M.*
AU  - Kobayashi, N.*
AU  - Ikushima, Y.M.*
AU  - Soeda, K.*
AU  - Tamura-Nakano, M.*
AU  - Muratani, M.*
AU  - Kobayashi, K.*
AU  - Blüher, M.
AU  - Brüning, J.C.*
AU  - Ueki, K.*
C1  - 64012
C2  - 52043
SP  - 680-689.e6
TI  - An antisense transcript transcribed from Irs2 locus contributes to the pathogenesis of hepatic steatosis in insulin resistance.
JO  - Cell Chem. Bio.
VL  - 29
IS  - 4
PY  - 2022
SN  - 2451-9448
ER  - 

TY  - JOUR
AB  - The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.
AU  - Napolitano, V.
AU  - Dabrowska, A.*
AU  - Schorpp, K.K.
AU  - Mourao, A.
AU  - Barreto-Duran, E.*
AU  - Benedyk, M.*
AU  - Botwina, P.*
AU  - Brandner, S.
AU  - Bostock, M.J.
AU  - Chykunova, Y.*
AU  - Czarna, A.*
AU  - Dubin, G.*
AU  - Fröhlich, T.
AU  - Hölscher, M.*
AU  - Jedrysik, M.*
AU  - Matsuda, A.*
AU  - Owczarek, K.*
AU  - Pachota, M.*
AU  - Plettenburg, O.
AU  - Potempa, J.S.*
AU  - Rothenaigner, I.
AU  - Schlauderer, F.
AU  - Slysz, K.*
AU  - Szczepanski, A.*
AU  - Greve-Isdahl Mohn, K.*
AU  - Blomberg, B.*
AU  - Sattler, M.
AU  - Hadian, K.
AU  - Popowicz, G.M.
AU  - Pyrc, K.*
C1  - 64075
C2  - 52076
SP  - 774-784.e8
TI  - Acriflavine, a clinically approved drug, inhibits SARS-CoV-2 and other betacoronaviruses.
JO  - Cell Chem. Bio.
VL  - 29
IS  - 5
PY  - 2022
SN  - 2451-9448
ER  - 

TY  - JOUR
AB  - Compounds that exhibit assay interference or undesirable mechanisms of bioactivity (“nuisance compounds”) are routinely encountered in cellular assays, including phenotypic and high-content screening assays. Much is known regarding compound-dependent assay interferences in cell-free assays. However, despite the essential role of cellular assays in chemical biology and drug discovery, there is considerably less known about nuisance compounds in more complex cell-based assays. In our view, a major obstacle to realizing the full potential of chemical biology will not just be difficult-to-drug targets or even the sheer number of targets, but rather nuisance compounds, due to their ability to waste significant resources and erode scientific trust. In this review, we summarize our collective academic, government, and industry experiences regarding cellular nuisance compounds. We describe assay design strategies to mitigate the impact of nuisance compounds and suggest best practices to efficiently address these compounds in complex biological settings. Nuisance compounds can waste significant resources by producing promising bioactivities that are attributable to undesirable mechanisms of action. Addressing nuisance compounds is particularly challenging in cellular assays. Dahlin et al. summarize academic, government, and industry experiences with assay design and hit triage to specifically address cellular nuisance compounds.
AU  - Dahlin, J.L.*
AU  - Auld, D.S.*
AU  - Rothenaigner, I.
AU  - Haney, S.*
AU  - Sexton, J.Z.*
AU  - Nissink, J.W.M.*
AU  - Walsh, J.*
AU  - Lee, J.A.*
AU  - Strelow, J.M.*
AU  - Willard, F.S.*
AU  - Ferrins, L.*
AU  - Baell, J.B.*
AU  - Walters, M.A.*
AU  - Hua, B.K.*
AU  - Hadian, K.
AU  - Wagner, B.K.*
C1  - 61425
C2  - 50240
CY  - 50 Hampshire St, Floor 5, Cambridge, Ma 02139 Usa
SP  - 356-370
TI  - Nuisance compounds in cellular assays.
JO  - Cell Chem. Bio.
VL  - 28
IS  - 3
PB  - Cell Press
PY  - 2021
SN  - 2451-9448
ER  - 

TY  - JOUR
AB  - The trace elements iron and selenium play decisive roles in a distinct form of necrotic cell death, known as ferroptosis. While iron promotes ferroptosis by contributing to Fenton-type reactions and uncontrolled lipid autoxidation, the hallmark of ferroptosis, selenium in the form of glutathione peroxidase 4 (GPX4), subdues phospholipid peroxidation and associated cell death. Beyond the canonical cystine/glutamate antiporter system x(c)(-)/glutathione/GPX4 nexus, recent studies unveiled the second mainstay in ferroptosis entailing extra-mitechondriai ubiquinone, ferroptosis suppressor protein 1, and NAD(P)H as electron donor. Unlike GPX4, this selenium- and thiol-independent system acts on the level of peroxyl radicals in membranes thereby restraining Hold peroxidation. Therefore, ferroptosis is a multifaceted cell-death paradigm characterized by several metabolic networks, whereby metabolic dyshomeostasis may cause ferroplotic cell death and organ failure. Here, we discuss the basic features of ferroptosis with a focus on selenium, offering exciting opportunities to control diseases linked to ferroptosis, including transient ischemia reperfusion and neurodegeneration.
AU  - Conrad, M.
AU  - Proneth, B.
C1  - 58845
C2  - 48505
CY  - 50 Hampshire St, Floor 5, Cambridge, Ma 02139 Usa
SP  - 409-419
TI  - Selenium: Tracing another essential element of ferroptotic cell death.
JO  - Cell Chem. Bio.
VL  - 27
IS  - 4
PB  - Cell Press
PY  - 2020
SN  - 2451-9448
ER  - 

TY  - JOUR
AB  - The methionine 1 (M1)-specific deubiquitinase (DUB) OTULIN acts as a negative regulator of nuclear factor κB signaling and immune homeostasis. By replacing Gly76 in distal ubiquitin (Ub) by dehydroalanine we designed the diubiquitin (diUb) activity-based probe UbG76Dha-Ub (OTULIN activity-based probe [ABP]) that couples to the catalytic site of OTULIN and thereby captures OTULIN in its active conformation. The OTULIN ABP displays high selectivity for OTULIN and does not label other M1-cleaving DUBs, including CYLD. The only detectable cross-reactivities were the labeling of USP5 (Isopeptidase T) and an ATP-dependent assembly of polyOTULIN ABP chains via Ub-activating E1 enzymes. Both cross-reactivities were abolished by the removal of the C-terminal Gly in the ABP's proximal Ub, yielding the specific OTULIN probe UbG76Dha-UbΔG76 (OTULIN ABPΔG76). Pull-downs demonstrate that substrate-bound OTULIN associates with the linear ubiquitin chain assembly complex (LUBAC). Thus, we present a highly selective ABP for OTULIN that will facilitate studying the cellular function of this essential DUB.
AU  - Weber, A.
AU  - Elliott, P.R.*
AU  - Pinto-Fernandez, A.*
AU  - Bonham, S.*
AU  - Kessler, B.M.*
AU  - Komander, D.*
AU  - El Oualid, F.*
AU  - Krappmann, D.
C1  - 51910
C2  - 43595
CY  - Cambridge
SP  - 1299-1313.e7
TI  - A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN.
JO  - Cell Chem. Bio.
VL  - 24
IS  - 10
PB  - Cell Press
PY  - 2017
SN  - 2451-9448
ER  - 

TY  - JOUR
AB  - Phenotypic drug discovery offers some advantages over target-based methods, mainly because it allows drug leads to be tested in systems that more closely model distinct disease states. However, a potential disadvantage is the difficulty of linking the observed phenotype to a specific cellular target. To address this problem, we developed DePick, a computational target de-convolution tool to determine targets specifically linked to small-molecule phenotypic screens. We applied DePick to eight publicly available screens and predicted 59 drug target-phenotype associations. In addition to literature-based evidence for our predictions, we provide experimental support for seven predicted associations. Interestingly, our analysis led to the discovery of a previously unrecognized connection between the Wnt signaling pathway and an aromatase, CYP19A1. These results demonstrate that the DePick approach can not only accelerate target de-convolution but also aid in discovery of new functionally relevant biological relationships.
AU  - Liu, X.
AU  - Baarsma, H.A.
AU  - Thiam, C.H.*
AU  - Montrone, C.
AU  - Brauner, B.
AU  - Fobo, G.
AU  - Heier, J.S.*
AU  - Duscha, S.
AU  - Königshoff, M.
AU  - Angeli, V.*
AU  - Ruepp, A.
AU  - Campillos, M.
C1  - 49562
C2  - 40791
CY  - Cambridge
SP  - 1302-1313
TI  - Systematic identification of pharmacological targets from small-molecule phenotypic screens.
JO  - Cell Chem. Bio.
VL  - 23
IS  - 10
PB  - Cell Press
PY  - 2016
SN  - 2451-9448
ER  -