Pub Date : 2024-03-25DOI: 10.1021/acschembio.4c00009
Drew V. Carson, Reecan J. Juarez, Truc Do, Zhongyue J. Yang and A. James Link*,
The development of new antimicrobial agents effective against Gram-negative bacteria remains a major challenge in drug discovery. The lasso peptide cloacaenodin has potent antimicrobial activity against multiple strains in the Enterobacter genus, one of the ESKAPE pathogens. Here, we show that cloacaenodin uses a previously uncharacterized TonB-dependent transporter, which we name CloU, to cross the outer membrane (OM) of susceptible bacteria. Inner membrane transport is mediated by the protein SbmA. CloU is distinct from the known OM transporters (FhuA and PupB) utilized by other antimicrobial lasso peptides and thus offers important insight into the spectrum of activity of cloacaenodin. Using knowledge of the transport pathway to predict other cloacaenodin-susceptible strains, we demonstrate the activity of cloacaenodin against clinical isolates of Enterobacter and of a Kluyvera strain. Further, we use molecular dynamics simulations and mutagenesis of CloU to explain the variation in cloacaenodin susceptibility observed across different strains of Enterobacter. This work expands the currently limited understanding of lasso peptide uptake and advances the potential of cloacaenodin as an antibiotic.
{"title":"Antimicrobial Lasso Peptide Cloacaenodin Utilizes a Unique TonB-Dependent Transporter to Access Susceptible Bacteria","authors":"Drew V. Carson, Reecan J. Juarez, Truc Do, Zhongyue J. Yang and A. James Link*, ","doi":"10.1021/acschembio.4c00009","DOIUrl":"10.1021/acschembio.4c00009","url":null,"abstract":"<p >The development of new antimicrobial agents effective against Gram-negative bacteria remains a major challenge in drug discovery. The lasso peptide cloacaenodin has potent antimicrobial activity against multiple strains in the <i>Enterobacter</i> genus, one of the ESKAPE pathogens. Here, we show that cloacaenodin uses a previously uncharacterized TonB-dependent transporter, which we name CloU, to cross the outer membrane (OM) of susceptible bacteria. Inner membrane transport is mediated by the protein SbmA. CloU is distinct from the known OM transporters (FhuA and PupB) utilized by other antimicrobial lasso peptides and thus offers important insight into the spectrum of activity of cloacaenodin. Using knowledge of the transport pathway to predict other cloacaenodin-susceptible strains, we demonstrate the activity of cloacaenodin against clinical isolates of <i>Enterobacter</i> and of a <i>Kluyvera</i> strain. Further, we use molecular dynamics simulations and mutagenesis of CloU to explain the variation in cloacaenodin susceptibility observed across different strains of <i>Enterobacter</i>. This work expands the currently limited understanding of lasso peptide uptake and advances the potential of cloacaenodin as an antibiotic.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140287584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-25DOI: 10.1021/acschembio.3c00803
Paige Dickson*,
The identification of novel covalent ligands for therapeutic purposes has long depended on serendipity, with dedicated hit finding techniques emerging only in the early 2000s. Advances in chemoproteomics have enabled robust characterization of putative drugs to derisk the unique liabilities associated with covalent hit molecules, leading to a renewed interest in this targeting modality. DNA-encoded library (DEL) technology has similarly emerged over the past two decades as a highly efficient method to identify new chemical equity toward protein targets of interest. A number of commercial and academic groups have reported methods in covalent DEL synthesis and hit identification; however, it is evident that there is still much to be done to fully realize the power of this technology for covalent ligand discovery. This perspective will explore the current approaches in covalent DEL technology and reflect on the next steps to advance this field.
长期以来,用于治疗目的的新型共价配体的鉴定一直依赖于偶然性,而专门的命中发现技术直到本世纪初才出现。化学蛋白质组学的进步使人们能够对可能的药物进行强有力的特征描述,从而发现与共价配体分子相关的独特缺陷,从而重新激发了人们对这种靶向方式的兴趣。DNA 编码文库(DEL)技术在过去二十年中同样崭露头角,成为一种高效的方法,用于鉴定针对感兴趣的蛋白质靶点的新化学权益。一些商业和学术团体已经报道了共价 DEL 合成和命中识别方法;然而,要充分发挥这项技术在共价配体发现方面的威力,显然还有许多工作要做。本视角将探讨共价 DEL 技术的现有方法,并思考推动这一领域发展的下一步措施。
{"title":"DNA-Encoded Library Technology─A Catalyst for Covalent Ligand Discovery","authors":"Paige Dickson*, ","doi":"10.1021/acschembio.3c00803","DOIUrl":"10.1021/acschembio.3c00803","url":null,"abstract":"<p >The identification of novel covalent ligands for therapeutic purposes has long depended on serendipity, with dedicated hit finding techniques emerging only in the early 2000s. Advances in chemoproteomics have enabled robust characterization of putative drugs to derisk the unique liabilities associated with covalent hit molecules, leading to a renewed interest in this targeting modality. DNA-encoded library (DEL) technology has similarly emerged over the past two decades as a highly efficient method to identify new chemical equity toward protein targets of interest. A number of commercial and academic groups have reported methods in covalent DEL synthesis and hit identification; however, it is evident that there is still much to be done to fully realize the power of this technology for covalent ligand discovery. This perspective will explore the current approaches in covalent DEL technology and reflect on the next steps to advance this field.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140287585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-22DOI: 10.1021/acschembio.4c00095
Lizett Ortiz de Ora, Julia M. Balsamo, Kylie S. Uyeda and Elizabeth N. Bess*,
Parkinson’s disease (PD) etiology is associated with aggregation and accumulation of α-synuclein (α-syn) proteins in midbrain dopaminergic neurons. Emerging evidence suggests that in certain subtypes of PD, α-syn aggregates originate in the gut and subsequently spread to the brain. However, mechanisms that instigate α-syn aggregation in the gut have remained elusive. In the brain, the aggregation of α-syn is induced by oxidized dopamine. Such a mechanism has not been explored in the context of the gastrointestinal tract, a niche harboring 46% of the body’s dopamine reservoirs. Here, we report that Enterobacteriaceae, a bacterial family prevalent in human gut microbiotas, induce α-syn aggregation. More specifically, our in vitro data indicate that respiration of nitrate by Escherichia coli K-12, which results in production of nitrite that mediates oxidation of Fe2+ to Fe3+, creates an oxidizing redox potential. These oxidizing conditions enabled the formation of dopamine-derived quinones and α-syn aggregates. Exposing nitrite, but not nitrate, to enteroendocrine STC-1 cells induced aggregation of α-syn that is natively expressed in these cells, which line the intestinal tract. Taken together, our findings indicate that bacterial nitrate reduction may be critical for initiating intestinal α-syn aggregation.
{"title":"Discovery of a Gut Bacterial Metabolic Pathway that Drives α-Synuclein Aggregation","authors":"Lizett Ortiz de Ora, Julia M. Balsamo, Kylie S. Uyeda and Elizabeth N. Bess*, ","doi":"10.1021/acschembio.4c00095","DOIUrl":"10.1021/acschembio.4c00095","url":null,"abstract":"<p >Parkinson’s disease (PD) etiology is associated with aggregation and accumulation of α-synuclein (α-syn) proteins in midbrain dopaminergic neurons. Emerging evidence suggests that in certain subtypes of PD, α-syn aggregates originate in the gut and subsequently spread to the brain. However, mechanisms that instigate α-syn aggregation in the gut have remained elusive. In the brain, the aggregation of α-syn is induced by oxidized dopamine. Such a mechanism has not been explored in the context of the gastrointestinal tract, a niche harboring 46% of the body’s dopamine reservoirs. Here, we report that <i>Enterobacteriaceae</i>, a bacterial family prevalent in human gut microbiotas, induce α-syn aggregation. More specifically, our <i>in vitro</i> data indicate that respiration of nitrate by <i>Escherichia coli</i> K-12, which results in production of nitrite that mediates oxidation of Fe<sup>2+</sup> to Fe<sup>3+</sup>, creates an oxidizing redox potential. These oxidizing conditions enabled the formation of dopamine-derived quinones and α-syn aggregates. Exposing nitrite, but not nitrate, to enteroendocrine STC-1 cells induced aggregation of α-syn that is natively expressed in these cells, which line the intestinal tract. Taken together, our findings indicate that bacterial nitrate reduction may be critical for initiating intestinal α-syn aggregation.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.4c00095","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140183065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-21DOI: 10.1021/acschembio.3c00798
Da Yeong Lee, Jonghwan Kim, Gyu Sung Lee, Sehwan Park, Jeongwon Song, Bum Soo Lee, Seoung Rak Lee, Ki Hyun Kim and Chung Sub Kim*,
In the field of natural product research, the rediscovery of already-known compounds is one of the significant issues hindering new drug development. Recently, an innovative approach called bioactivity-HiTES has been developed to overcome this limitation, and several new bioactive metabolites have been successfully characterized by this method. In this study, we applied bioactivity-HiTES to Corynebacterium matruchotii, the human oral bacterium, with 3120 clinical drugs as potential elicitors. As a result, we identified two cryptic metabolites, methylindole-3-acetate (MIAA) and indole-3-acetic acid (IAA), elicited by imidafenacin, a urinary antispasmodic drug approved by the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). MIAA showed weak antibacterial activity against a pulmonary disease-causing Mycobacterium conceptionense with an IC50 value of 185.7 μM. Unexpectedly, we also found that C. matruchotii metabolized fludarabine phosphate, a USFDA-approved anticancer drug, to 2-fluoroadenine which displayed moderate antibacterial activity against both Bacillus subtilis and Escherichia coli, with IC50 values of 8.9 and 20.1 μM, respectively. Finally, acelarin, a prodrug of the anticancer drug gemcitabine, was found to exhibit unreported antibacterial activity against B. subtilis with an IC50 value of 33.6 μM through the bioactivity-HiTES method as well. These results indicate that bioactivity-HiTES can also be applied to discover biotransformed products in addition to finding cryptic metabolites in microbes.
{"title":"Characterization of Chemical Interactions between Clinical Drugs and the Oral Bacterium, Corynebacterium matruchotii, via Bioactivity-HiTES","authors":"Da Yeong Lee, Jonghwan Kim, Gyu Sung Lee, Sehwan Park, Jeongwon Song, Bum Soo Lee, Seoung Rak Lee, Ki Hyun Kim and Chung Sub Kim*, ","doi":"10.1021/acschembio.3c00798","DOIUrl":"10.1021/acschembio.3c00798","url":null,"abstract":"<p >In the field of natural product research, the rediscovery of already-known compounds is one of the significant issues hindering new drug development. Recently, an innovative approach called bioactivity-HiTES has been developed to overcome this limitation, and several new bioactive metabolites have been successfully characterized by this method. In this study, we applied bioactivity-HiTES to <i>Corynebacterium matruchotii</i>, the human oral bacterium, with 3120 clinical drugs as potential elicitors. As a result, we identified two cryptic metabolites, methylindole-3-acetate (MIAA) and indole-3-acetic acid (IAA), elicited by imidafenacin, a urinary antispasmodic drug approved by the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). MIAA showed weak antibacterial activity against a pulmonary disease-causing <i>Mycobacterium conceptionense</i> with an IC<sub>50</sub> value of 185.7 μM. Unexpectedly, we also found that <i>C. matruchotii</i> metabolized fludarabine phosphate, a USFDA-approved anticancer drug, to 2-fluoroadenine which displayed moderate antibacterial activity against both <i>Bacillus subtilis</i> and <i>Escherichia coli</i>, with IC<sub>50</sub> values of 8.9 and 20.1 μM, respectively. Finally, acelarin, a prodrug of the anticancer drug gemcitabine, was found to exhibit unreported antibacterial activity against <i>B. subtilis</i> with an IC<sub>50</sub> value of 33.6 μM through the bioactivity-HiTES method as well. These results indicate that bioactivity-HiTES can also be applied to discover biotransformed products in addition to finding cryptic metabolites in microbes.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140183063","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-21DOI: 10.1021/acschembio.3c00777
Heng Zhao, Praveen Kumar, Tiago Jose Paschoal Sobreira, Mackenzie Smith, Steven Novick, Anders Johansson, Anna Luchniak, Andrew Zhang, Kevin J Woollard, Niklas Larsson* and Aarti Kawatkar*,
Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and selective small-molecule inhibitor of the NLRP3 pathway and has been validated in numerous species and disease models. Although the capacity of MCC950 to block NLRP3 signaling is well-established, it is still critical to identify the mechanism of action and molecular targets of MCC950 to inform and derisk drug development. Quantitative proteomics performed in disease-relevant systems provides a powerful method to study both direct and indirect pharmacological responses to small molecules to elucidate the mechanism of action and confirm target engagement. A comprehensive target deconvolution campaign requires the use of complementary chemical biology techniques. Here we applied two orthogonal chemical biology techniques: compressed Cellular Thermal Shift Assay (CETSA) and photoaffinity labeling chemoproteomics, performed under biologically relevant conditions with LPS-primed THP-1 cells, thereby deconvoluting, for the first time, the molecular targets of MCC950 using chemical biology techniques. In-cell chemoproteomics with inlysate CETSA confirmed the suspected mechanism as the disruption of inflammasome formation via NLRP3. Further cCETSA (c indicates compressed) in live cells mapped the stabilization of NLRP3 inflammasome pathway proteins, highlighting modulation of the targeted pathway. This is the first evidence of direct MCC950 engagement with endogenous NLRP3 in a human macrophage cellular system using discovery proteomics chemical biology techniques, providing critical information for inflammasome studies.
{"title":"Integrated Proteomics Characterization of NLRP3 Inflammasome Inhibitor MCC950 in Monocytic Cell Line Confirms Direct MCC950 Engagement with Endogenous NLRP3","authors":"Heng Zhao, Praveen Kumar, Tiago Jose Paschoal Sobreira, Mackenzie Smith, Steven Novick, Anders Johansson, Anna Luchniak, Andrew Zhang, Kevin J Woollard, Niklas Larsson* and Aarti Kawatkar*, ","doi":"10.1021/acschembio.3c00777","DOIUrl":"10.1021/acschembio.3c00777","url":null,"abstract":"<p >Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and selective small-molecule inhibitor of the NLRP3 pathway and has been validated in numerous species and disease models. Although the capacity of MCC950 to block NLRP3 signaling is well-established, it is still critical to identify the mechanism of action and molecular targets of MCC950 to inform and derisk drug development. Quantitative proteomics performed in disease-relevant systems provides a powerful method to study both direct and indirect pharmacological responses to small molecules to elucidate the mechanism of action and confirm target engagement. A comprehensive target deconvolution campaign requires the use of complementary chemical biology techniques. Here we applied two orthogonal chemical biology techniques: compressed Cellular Thermal Shift Assay (CETSA) and photoaffinity labeling chemoproteomics, performed under biologically relevant conditions with LPS-primed THP-1 cells, thereby deconvoluting, for the first time, the molecular targets of MCC950 using chemical biology techniques. In-cell chemoproteomics with inlysate CETSA confirmed the suspected mechanism as the disruption of inflammasome formation via NLRP3. Further cCETSA (c indicates compressed) in live cells mapped the stabilization of NLRP3 inflammasome pathway proteins, highlighting modulation of the targeted pathway. This is the first evidence of direct MCC950 engagement with endogenous NLRP3 in a human macrophage cellular system using discovery proteomics chemical biology techniques, providing critical information for inflammasome studies.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140173524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nonreceptor tyrosine kinase c-Src plays a crucial role in cell signaling and contributes to tumor progression. However, the development of selective c-Src inhibitors turns out to be challenging. In our previous study, we performed posttranslational modification-inspired drug design (PTMI-DD) to provide a plausible way for designing selective kinase inhibitors. In this study, after identifying a unique pocket comprising a less conserved cysteine and an autophosphorylation site in c-Src as well as a promiscuous covalent inhibitor, chemical optimization was performed to obtain (R)-LW-Srci-8 with nearly 75-fold improved potency (IC50 = 35.83 ± 7.21 nM). Crystallographic studies revealed the critical C–F···C═O interactions that may contribute to tight binding. The kinact and Ki values validated the improved binding affinity and decreased warhead reactivity of (R)-LW-Srci-8 for c-Src. Notably, in vitro tyrosine kinase profiling and cellular activity-based protein profiling (ABPP) cooperatively indicated a specific inhibition of c-Src by (R)-LW-Srci-8. Intriguingly, (R)-LW-Srci-8 preferentially binds to inactive c-Src with unphosphorylated Y419 both in vitro and in cells, subsequently disrupting the autophosphorylation. Collectively, our study demonstrated the feasibility of developing selective kinase inhibitors by cotargeting a nucleophilic residue and a posttranslational modification site and providing a chemical probe for c-Src functional studies.
{"title":"Discovery of a Covalent Inhibitor Selectively Targeting the Autophosphorylation Site of c-Src Kinase","authors":"Huimin Zhang, Dounan Xu, Hongchan Huang, Hao Jiang, Linghao Hu, Liping Liu, Ge Sun, Jing Gao, Yuanqing Li, Cuicui Xia, Shijie Chen, Hu Zhou, Xiangqian Kong*, Mingliang Wang* and Cheng Luo*, ","doi":"10.1021/acschembio.4c00048","DOIUrl":"10.1021/acschembio.4c00048","url":null,"abstract":"<p >Nonreceptor tyrosine kinase c-Src plays a crucial role in cell signaling and contributes to tumor progression. However, the development of selective c-Src inhibitors turns out to be challenging. In our previous study, we performed posttranslational modification-inspired drug design (PTMI-DD) to provide a plausible way for designing selective kinase inhibitors. In this study, after identifying a unique pocket comprising a less conserved cysteine and an autophosphorylation site in c-Src as well as a promiscuous covalent inhibitor, chemical optimization was performed to obtain <b>(</b><i><b>R</b></i><b>)-LW-Srci-8</b> with nearly 75-fold improved potency (IC<sub>50</sub> = 35.83 ± 7.21 nM). Crystallographic studies revealed the critical C–F···C═O interactions that may contribute to tight binding. The <i>k</i><sub>inact</sub> and <i>K<sub>i</sub></i> values validated the improved binding affinity and decreased warhead reactivity of <b>(</b><i><b>R</b></i><b>)-LW-Srci-8</b> for c-Src. Notably, in vitro tyrosine kinase profiling and cellular activity-based protein profiling (ABPP) cooperatively indicated a specific inhibition of c-Src by <b>(</b><i><b>R</b></i><b>)-LW-Srci-8</b>. Intriguingly, <b>(</b><i><b>R</b></i><b>)-LW-Srci-8</b> preferentially binds to inactive c-Src with unphosphorylated Y419 both in vitro and in cells, subsequently disrupting the autophosphorylation. Collectively, our study demonstrated the feasibility of developing selective kinase inhibitors by cotargeting a nucleophilic residue and a posttranslational modification site and providing a chemical probe for c-Src functional studies.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140183064","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-20DOI: 10.1021/acschembio.3c00699
Ralston B. Goldfarb, Matthew J. Atala Pleshinger, David F. Yan and Drew J. Adams*,
Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimic the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example, on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provide more realistic levels of lipids. Here, we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. The mechanism of action studies indicates that ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets delta 4-desaturase, sphingolipid 1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies suggest ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and reveal a general screening strategy for identifying additional cancer dependencies masked by the superabundance of medium lipids.
{"title":"Lipid-Restricted Culture Media Reveal Unexpected Cancer Cell Sensitivities","authors":"Ralston B. Goldfarb, Matthew J. Atala Pleshinger, David F. Yan and Drew J. Adams*, ","doi":"10.1021/acschembio.3c00699","DOIUrl":"10.1021/acschembio.3c00699","url":null,"abstract":"<p >Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimic the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example, on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provide more realistic levels of lipids. Here, we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. The mechanism of action studies indicates that ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets delta 4-desaturase, sphingolipid 1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies suggest ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and reveal a general screening strategy for identifying additional cancer dependencies masked by the superabundance of medium lipids.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140173525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-16DOI: 10.1021/acschembio.3c00728
Yu Zhang, Zhe Yang, Dilizhatai Saimi, Xiaowen Shen, Junqing Ye, Bingke Yu, Noah Pefaur, Justin M. Scheer, Andrew E. Nixon* and Zhixing Chen*,
Bispecific antibodies (BsAbs) represent an emerging class of biologics that can recognize two different antigens or epitopes. T-cell engagers (TcEs) bind two targets in trans on the cell surface of the effector and target cell to induce proximal immune effects, opening exciting windows for immunotherapies. To date, the engineering of BsAbs has been mainly focused on tuning the molecular weight and valency. However, the effects of spatial factors on the biological functions of BsAbs have been less explored due to the lack of biochemical methods to precisely manipulate protein geometry. Here, we studied the geometric effects of the TcEs. First, by genetically inserting rigidly designed ankyrin repeat proteins into TcEs, we revealed that the efficacy progressively decreased as the spacer distance of the two binding domains increased. Then, we constructed 26 pairs of TcEs with the same size but varying orientations using click chemistry-mediated conjugation at different mutation sites. We found that linear ligation sites play a minor role in modulating cell-killing efficacy. Next, we rendered the TcEs’ advanced topology by cyclization chemistry using the SpyTag/SpyCatcher pair or sortase ligation approaches. Cyclized TcEs were generally more potent than their linear counterparts. Particularly, sortase A cyclized TcEs, bearing a minimal tagging motif, exhibited better cell-killing efficacy in vitro and improved stability both in vitro and in vivo compared to the linear TcE. This work combines modern bioconjugation chemistry and protein engineering tools for antibody engineering, shedding light on the elusive spatial factors of BsAbs functionality.
{"title":"Geometric Antibody Engineering Reveals the Spatial Factor on the Efficacy of Bispecific T Cell Engagers","authors":"Yu Zhang, Zhe Yang, Dilizhatai Saimi, Xiaowen Shen, Junqing Ye, Bingke Yu, Noah Pefaur, Justin M. Scheer, Andrew E. Nixon* and Zhixing Chen*, ","doi":"10.1021/acschembio.3c00728","DOIUrl":"10.1021/acschembio.3c00728","url":null,"abstract":"<p >Bispecific antibodies (BsAbs) represent an emerging class of biologics that can recognize two different antigens or epitopes. T-cell engagers (TcEs) bind two targets in trans on the cell surface of the effector and target cell to induce proximal immune effects, opening exciting windows for immunotherapies. To date, the engineering of BsAbs has been mainly focused on tuning the molecular weight and valency. However, the effects of spatial factors on the biological functions of BsAbs have been less explored due to the lack of biochemical methods to precisely manipulate protein geometry. Here, we studied the geometric effects of the TcEs. First, by genetically inserting rigidly designed ankyrin repeat proteins into TcEs, we revealed that the efficacy progressively decreased as the spacer distance of the two binding domains increased. Then, we constructed 26 pairs of TcEs with the same size but varying orientations using click chemistry-mediated conjugation at different mutation sites. We found that linear ligation sites play a minor role in modulating cell-killing efficacy. Next, we rendered the TcEs’ advanced topology by cyclization chemistry using the SpyTag/SpyCatcher pair or sortase ligation approaches. Cyclized TcEs were generally more potent than their linear counterparts. Particularly, sortase A cyclized TcEs, bearing a minimal tagging motif, exhibited better cell-killing efficacy in vitro and improved stability both in vitro and in vivo compared to the linear TcE. This work combines modern bioconjugation chemistry and protein engineering tools for antibody engineering, shedding light on the elusive spatial factors of BsAbs functionality.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140139593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-14DOI: 10.1021/acschembio.3c00607
Paul D. O’Dowd, Andres S. Guerrero, Katelyn R. Alley, Hannah C. Pigg, Fiona O’Neill, Justine Meiller, Chloe Hobbs, Daniel A. Rodrigues, Brendan Twamley, Finbarr O’Sullivan, Victoria J. DeRose and Darren M. Griffith*,
It is well established that oxaliplatin, one of the three Pt(II) anticancer drugs approved worldwide, and phenanthriplatin, an important preclinical monofunctional Pt(II) anticancer drug, possess a different mode of action from that of cisplatin and carboplatin, namely, the induction of nucleolar stress. The exact mechanisms that lead to Pt-induced nucleolar stress are, however, still poorly understood. As such, studies aimed at better understanding the biological targets of both oxaliplatin and phenanthriplatin are urgently needed to expand our understanding of Pt-induced nucleolar stress and guide the future design of Pt chemotherapeutics. One approach that has seen great success in the past is the use of Pt-click complexes to study the biological targets of Pt drugs. Herein, we report the synthesis and characterization of the first examples of click-capable phenanthriplatin complexes. Furthermore, through monitoring the relocalization of nucleolar proteins, RNA transcription levels, and DNA damage repair biomarker γH2AX, and by investigating their in vitro cytotoxicity, we show that these complexes successfully mimic the cellular responses observed for phenanthriplatin treatment in the same experiments. The click-capable phenanthriplatin derivatives described here expand the existing library of Pt-click complexes. Significantly they are suitable for studying nucleolar stress mechanisms and further elucidating the biological targets of Pt complexes.
{"title":"Click-Capable Phenanthriplatin Derivatives as Tools to Study Pt(II)-Induced Nucleolar Stress","authors":"Paul D. O’Dowd, Andres S. Guerrero, Katelyn R. Alley, Hannah C. Pigg, Fiona O’Neill, Justine Meiller, Chloe Hobbs, Daniel A. Rodrigues, Brendan Twamley, Finbarr O’Sullivan, Victoria J. DeRose and Darren M. Griffith*, ","doi":"10.1021/acschembio.3c00607","DOIUrl":"10.1021/acschembio.3c00607","url":null,"abstract":"<p >It is well established that oxaliplatin, one of the three Pt(II) anticancer drugs approved worldwide, and phenanthriplatin, an important preclinical monofunctional Pt(II) anticancer drug, possess a different mode of action from that of cisplatin and carboplatin, namely, the induction of nucleolar stress. The exact mechanisms that lead to Pt-induced nucleolar stress are, however, still poorly understood. As such, studies aimed at better understanding the biological targets of both oxaliplatin and phenanthriplatin are urgently needed to expand our understanding of Pt-induced nucleolar stress and guide the future design of Pt chemotherapeutics. One approach that has seen great success in the past is the use of Pt-click complexes to study the biological targets of Pt drugs. Herein, we report the synthesis and characterization of the first examples of click-capable phenanthriplatin complexes. Furthermore, through monitoring the relocalization of nucleolar proteins, RNA transcription levels, and DNA damage repair biomarker γH2AX, and by investigating their <i>in vitro</i> cytotoxicity, we show that these complexes successfully mimic the cellular responses observed for phenanthriplatin treatment in the same experiments. The click-capable phenanthriplatin derivatives described here expand the existing library of Pt-click complexes. Significantly they are suitable for studying nucleolar stress mechanisms and further elucidating the biological targets of Pt complexes.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.3c00607","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140118104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-13DOI: 10.1021/acschembio.3c00695
Theodore J. Litberg, and , Scott Horowitz*,
The role of nucleic acids in protein folding and aggregation is an area of continued research, with relevance to understanding both basic biological processes and disease. In this review, we provide an overview of the trajectory of research on both nucleic acids as chaperones and their roles in several protein misfolding diseases. We highlight key questions that remain on the biophysical and biochemical specifics of how nucleic acids have large effects on multiple proteins’ folding and aggregation behavior and how this pertains to multiple protein misfolding diseases.
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