SLAS Discovery最新文献

{"title":"Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications","authors":"Shitalben Patel ,&nbsp;Marie Karlsson ,&nbsp;Joseph T. Klahn ,&nbsp;Frank Gambino Jr. ,&nbsp;Helena Costa ,&nbsp;Kathleen A. McGuire ,&nbsp;Christina K. Baumgartner ,&nbsp;Jon Williams ,&nbsp;Sarah Sandoz ,&nbsp;James E. Kath","doi":"10.1016/j.slasd.2023.12.007","DOIUrl":"10.1016/j.slasd.2023.12.007","url":null,"abstract":"<div><p>The cellular thermal shift assay (CETSA®) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA® has been used for semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based readouts using cell lines. However, there has been no quantitative evaluation of CETSA® in unprocessed human whole blood, which is preferred for clinical applications. Here we report two separate assay formats – Alpha CETSA® and MSD CETSA® – that require less than 100 μL of whole blood per sample without PBMC isolation. We chose RIPK1 as a proof-of-concept target and, by measuring engagement of seven different inhibitors, demonstrate high assay sensitivity and robustness. These quantitative CETSA® platforms enable possible applications in preclinical pharmacokinetic-pharmacodynamic studies, and direct target engagement with small molecules in clinical trials.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 2","pages":"Article 100135"},"PeriodicalIF":3.1,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555223000977/pdfft?md5=c695ee332c2a10c9bcaef4c168483824&pid=1-s2.0-S2472555223000977-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138693365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Screening approaches for the identification of Nrf2-Keap1 protein-protein interaction inhibitors targeting hot spot residues","authors":"Wataru Asano ,&nbsp;Rie Hantani ,&nbsp;Toru Uhara ,&nbsp;François Debaene ,&nbsp;Akihiro Nomura ,&nbsp;Keishi Yamaguchi ,&nbsp;Tsuyoshi Adachi ,&nbsp;Kazuki Otake ,&nbsp;Kazuhito Harada ,&nbsp;Yoshiji Hantani","doi":"10.1016/j.slasd.2023.11.001","DOIUrl":"10.1016/j.slasd.2023.11.001","url":null,"abstract":"<div><p>Protein-protein interactions (PPIs) play a crucial role in most biological processes and are important targets in the development of therapeutic agents. However, small molecule drug discovery that targets PPIs remains very challenging. Targeting hot spot residues is considered the best option for inhibiting such interactions, but there are few examples of how knowledge of hot spots can be used in high throughput screening to find hit compounds. A substrate adaptor protein for a ubiquitin ligase complex, Kelch-like ECH-associated protein 1 (Keap1), negatively modulates the expression of genes involved in cellular protection against oxidative stress. Here, we focused on three arginine hot spot residues in the Keap1 substrate binding pocket (Arg380, Arg415, and Arg483), and screened the carboxylic acid library owned by Japan Tobacco Inc. for compounds that interact with the arginine residues in differential scanning fluorescence assays. Furthermore, we identified several small molecule compounds that specifically bind to the Keap1 Kelch domain hot spots by comparing binding to alanine mutant proteins (R380A, R415A, and R483A) with binding to the wild-type protein using surface plasmon resonance (SPR) screening. These compounds inhibited the protein-protein interaction between the Keap1 Kelch domain and the nuclear factor erythroid 2-related factor 2 (Nrf2) peptide, and the ubiquitination of Nrf2 catalyzed by the Cul3/RINGBox 1 E3 ligase. In addition, the binding mode of one compound (Compound 4) was determined by X-ray crystallography after validation of binding by isothermal titration calorimetry, native mass spectrometry, and nuclear magnetic resonance. Compound 4 had favorable thermodynamic properties, and noncovalently bound to Keap1 with a stoichiometry of 1:1. Our results suggest that Compound 4 could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions such as oxidative stress response, inflammation, and carcinogenesis. We believe that the use of a set of complementary biophysical techniques including the SPR assay with single alanine mutant of hot spots provides opportunities to identify hit compounds for developing inhibitors of PPIs.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 2","pages":"Article 100125"},"PeriodicalIF":3.1,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555223000795/pdfft?md5=15c038b4f2ad2614e505129c72ef0593&pid=1-s2.0-S2472555223000795-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71489819","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ex vivo discovery of synergistic drug combinations for hematologic malignancies","authors":"Kamran A. Ali ,&nbsp;Reecha D. Shah ,&nbsp;Anukriti Dhar ,&nbsp;Nina M. Myers ,&nbsp;Cameron Nguyen ,&nbsp;Arisa Paul ,&nbsp;Jordan E. Mancuso ,&nbsp;A. Scott Patterson ,&nbsp;James P. Brody ,&nbsp;Diane Heiser","doi":"10.1016/j.slasd.2023.12.001","DOIUrl":"10.1016/j.slasd.2023.12.001","url":null,"abstract":"<div><p>Combination therapies have improved outcomes for patients with acute myeloid leukemia (AML). However, these patients still have poor overall survival. Although many combination therapies are identified with high-throughput screening (HTS), these approaches are constrained to disease models that can be grown in large volumes (e.g., immortalized cell lines), which have limited translational utility. To identify more effective and personalized treatments, we need better strategies for screening and exploring potential combination therapies. Our objective was to develop an HTS platform for identifying effective combination therapies with highly translatable ex vivo disease models that use size-limited, primary samples from patients with leukemia (AML and myelodysplastic syndrome). We developed a system, ComboFlow, that comprises three main components: MiniFlow, ComboPooler, and AutoGater. MiniFlow conducts ex vivo drug screening with a miniaturized flow-cytometry assay that uses minimal amounts of patient sample to maximize throughput. ComboPooler incorporates computational methods to design efficient screens of pooled drug combinations. AutoGater is an automated gating classifier for flow cytometry that uses machine learning to rapidly analyze the large datasets generated by the assay. We used ComboFlow to efficiently screen more than 3000 drug combinations across 20 patient samples using only 6 million cells per patient sample. In this screen, ComboFlow identified the known synergistic combination of bortezomib and panobinostat. ComboFlow also identified a novel drug combination, dactinomycin and fludarabine, that synergistically killed leukemic cells in 35 % of AML samples. This combination also had limited effects in normal, hematopoietic progenitors. In conclusion, ComboFlow enables exploration of massive landscapes of drug combinations that were previously inaccessible in ex vivo models. We envision that ComboFlow can be used to discover more effective and personalized combination therapies for cancers amenable to ex vivo models.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 2","pages":"Article 100129"},"PeriodicalIF":3.1,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555223000849/pdfft?md5=456103813a56aee8d81306efba4130ea&pid=1-s2.0-S2472555223000849-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138693366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Screening for molecular glues – Challenges and opportunities","authors":"Geoffrey A. Holdgate ,&nbsp;Catherine Bardelle ,&nbsp;Sophia K. Berry ,&nbsp;Alice Lanne ,&nbsp;Maria Emanuela Cuomo","doi":"10.1016/j.slasd.2023.12.008","DOIUrl":"10.1016/j.slasd.2023.12.008","url":null,"abstract":"<div><p>Molecular glues are small molecules, typically smaller than PROTACs, and usually with improved physicochemical properties that aim to stabilise the interaction between two proteins. Most often this approach is used to improve or induce an interaction between the target and an E3 ligase, but other interactions which stabilise interactions to increase activity or to inhibit binding to a natural effector have also been demonstrated. This review will describe the effects of induced proximity, discuss current methods used to identify molecular glues and introduce approaches that could be adapted for molecular glue screening.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 2","pages":"Article 100136"},"PeriodicalIF":3.1,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555223000989/pdfft?md5=c74d6c64cf64b59b7ca21ecacd28d996&pid=1-s2.0-S2472555223000989-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138693358","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Memoriam","authors":"","doi":"10.1016/j.slasd.2023.05.002","DOIUrl":"10.1016/j.slasd.2023.05.002","url":null,"abstract":"","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 2","pages":"Article 100099"},"PeriodicalIF":3.1,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555223000370/pdfft?md5=a06c991e1487d4ea02a53c1850dfad55&pid=1-s2.0-S2472555223000370-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134903291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a high-throughput screening platform to identify new therapeutic agents for Medulloblastoma Group 3","authors":"Inés Fallon ,&nbsp;Henar Hernando ,&nbsp;Olga Almacellas-Rabaiget ,&nbsp;Berta Marti-Fuster ,&nbsp;Cesare Spadoni ,&nbsp;Darell D Bigner ,&nbsp;Eva Méndez","doi":"10.1016/j.slasd.2024.100147","DOIUrl":"10.1016/j.slasd.2024.100147","url":null,"abstract":"<div><p>Pediatric brain tumors (PBTs) represent about 25 % of all pediatric cancers and are the most common solid tumors in children and adolescents. Medulloblastoma (MB) is the most frequently occurring malignant PBT, accounting for almost 10 % of all pediatric cancer deaths. MB Group 3 (MB G3) accounts for 25–30 % of all MB cases and has the worst outcome, particularly when associated with MYC amplification. However, no targeted treatments for this group have been developed so far.</p><p>Here we describe a unique high throughput screening (HTS) platform specifically designed to identify new therapies for MB G3. The platform incorporates optimized and validated 2D and 3D efficacy and toxicity models, that account for tumor heterogenicity, limited efficacy and unacceptable toxicity from the very early stage of drug discovery. The platform has been validated by conducting a pilot HTS campaign with a 1280 lead-like compound library. Results showed 8 active compounds, targeting MB reported targets and several are currently approved or in clinical trials for pediatric patients with PBTs, including MB. Moreover, hits were combined to avoid tumor resistance, identifying 3 synergistic pairs, one of which is currently under clinical study for recurrent MB and other PBTs.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 2","pages":"Article 100147"},"PeriodicalIF":3.1,"publicationDate":"2024-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000091/pdfft?md5=23b3e2054d9f15dc299480ee9099b87f&pid=1-s2.0-S2472555224000091-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139736889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The openOCHEM consensus model is the best-performing open-source predictive model in the First EUOS/SLAS joint compound solubility challenge","authors":"Andrea Hunklinger ,&nbsp;Peter Hartog ,&nbsp;Martin Šícho ,&nbsp;Guillaume Godin ,&nbsp;Igor V. Tetko","doi":"10.1016/j.slasd.2024.01.005","DOIUrl":"10.1016/j.slasd.2024.01.005","url":null,"abstract":"<div><p>The EUOS/SLAS challenge aimed to facilitate the development of reliable algorithms to predict the aqueous solubility of small molecules using experimental data from 100 K compounds. In total, hundred teams took part in the challenge to predict low, medium and highly soluble compounds as measured by the nephelometry assay. This article describes the winning model, which was developed using the publicly available Online CHEmical database and Modeling environment (OCHEM) available on the website <span>https://ochem.eu/article/27</span><svg><path></path></svg>. We describe in detail the assumptions and steps used to select methods, descriptors and strategy which contributed to the winning solution. In particular we show that consensus based on 28 models calculated using descriptor-based and representation learning methods allowed us to obtain the best score, which was higher than those based on individual approaches or consensus models developed using each individual approach. A combination of diverse models allowed us to decrease both bias and variance of individual models and to calculate the highest score. The model based on Transformer CNN contributed the best individual score thus highlighting the power of Natural Language Processing (NLP) methods. The inclusion of information about aleatoric uncertainty would be important to better understand and use the challenge data by the contestants.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 2","pages":"Article 100144"},"PeriodicalIF":3.1,"publicationDate":"2024-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000066/pdfft?md5=6b7aa512858162a77178db862a6715d1&pid=1-s2.0-S2472555224000066-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139678098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Perspectives on phenotypic screening−Screen Design and Assay Technology Special Interest Group","authors":"Chorom Pak ,&nbsp;Kaylene J. Simpson ,&nbsp;Andrea D. Weston ,&nbsp;Mary Ellen Cvijic ,&nbsp;Kenda Evans ,&nbsp;Andrew D. Napper","doi":"10.1016/j.slasd.2024.02.001","DOIUrl":"10.1016/j.slasd.2024.02.001","url":null,"abstract":"<div><p>Here we offer perspectives on phenotypic screening based on a wide-ranging discussion entitled “Phenotypic screening, target ID, and multi-omics: enabling more disease relevance in early discovery?” at the Screen Design and Assay Technology Special Interest Group Meeting at the 2023 SLAS Conference. During the session, the authors shared their own experience from within their respective organizations, followed by an open discussion with the audience. It was recognized that while substantial progress has been made towards translating disease-relevant phenotypic early discovery into clinical success, there remain significant operational and scientific challenges to implementing phenotypic screening efforts, and improving translation of screening hits comes with substantial resource demands and organizational commitment. This Perspective assesses progress, highlights pitfalls, and offers possible solutions to help unlock the therapeutic potential of phenotypic drug discovery. Areas explored comprise screening and hit validation strategy, choice of cellular model, moving beyond 2D cell culture into three dimensions, and leveraging high-dimensional data sets downstream of phenotypic screens.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 2","pages":"Article 100146"},"PeriodicalIF":3.1,"publicationDate":"2024-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S247255522400008X/pdfft?md5=6c8f5d753c3f54536e16525d3c3ab6e0&pid=1-s2.0-S247255522400008X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139678102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Activity and inhibition of the SARS-CoV-2 Omicron nsp13 R392C variant using RNA duplex unwinding assays","authors":"Nicole L. Inniss ,&nbsp;Margarita Rzhetskaya ,&nbsp;Ted Ling-Hu ,&nbsp;Ramon Lorenzo-Redondo ,&nbsp;Kelly E. Bachta ,&nbsp;Karla J.F. Satchell ,&nbsp;Judd F. Hultquist","doi":"10.1016/j.slasd.2024.01.006","DOIUrl":"10.1016/j.slasd.2024.01.006","url":null,"abstract":"<div><p>SARS-CoV-2 nsp13 helicase is an essential enzyme for viral replication and a promising target for antiviral drug development. This study compares the double-stranded RNA (dsRNA) unwinding activity of nsp13 and the Omicron nsp13^R392C variant, which is predominant in currently circulating lineages. Using <em>in vitro</em> gel- and fluorescence-based assays, we found that both nsp13 and nsp13^R392C have dsRNA unwinding activity with equivalent kinetics. Furthermore, the R392C mutation had no effect on the efficiency of the nsp13-specific helicase inhibitor SSYA10-001. We additionally confirmed the activity of several other helicase inhibitors against nsp13, including punicalagin that inhibited dsRNA unwinding at nanomolar concentrations. Overall, this study reveals the utility of using dsRNA unwinding assays to screen small molecules for antiviral activity against nsp13 and the Omicron nsp13^R392C variant. Continual monitoring of newly emergent variants will be essential for considering resistance profiles of lead compounds as they are advanced towards next-generation therapeutic development.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 3","pages":"Article 100145"},"PeriodicalIF":3.1,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000078/pdfft?md5=f7dc6d1a4af32c1d663782fff14897d5&pid=1-s2.0-S2472555224000078-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139673776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons","authors":"Zhi-Bin Tong,&nbsp;Ruili Huang,&nbsp;John Braisted,&nbsp;Pei-Hsuan Chu,&nbsp;Anton Simeonov,&nbsp;David L. Gerhold","doi":"10.1016/j.slasd.2024.01.004","DOIUrl":"10.1016/j.slasd.2024.01.004","url":null,"abstract":"<div><p>Three-dimensional (3D) cell culture <em>in vitro</em> promises to improve representation of neuron physiology <em>in vivo</em>. This inspired development of a 3D culture platform for LUHMES (Lund Human Mesencephalic) dopaminergic neurons for high-throughput screening (HTS) of chemicals for neurotoxicity. Three culture platforms, adhesion (2D-monolayer), 3D-suspension, and 3D-shaken, were compared to monitor mRNA expression of seven neuronal marker genes, <em>DCX, DRD2, ENO2, NEUROD4, SYN1, TH,</em> and <em>TUBB3</em>. These seven marker genes reached similar maxima in all three formats, with the two 3D platforms showing similar kinetics, whereas several markers peaked earlier in 2D adhesion compared to both 3D culture platforms. The differentiated LUHMES (dLUHMES) neurons treated with ziram, methylmercury or thiram dynamically increased expression of metallothionein biomarker genes <em>MT1G, MT1E</em> and <em>MT2A</em> at 6 h. These gene expression increases were generally more dynamic in 2D adhesion cultures than in 3D cultures, but were generally comparable between 3D-suspension and 3D-u plate (low binding) platforms. Finally, we adapted 3D-suspension culture of dLUHMES and neural stem cells to 1536 well plates with a HTS cytotoxicity assay. This HTS assay revealed that cytotoxicity IC₅₀ values were not significantly different between adhesion and 3D-suspension platforms for 31 of 34 (91%) neurotoxicants tested, whereas IC₅₀ values were significantly different for at least two toxicants. In summary, the 3D-suspension culture platform for LUHMES dopaminergic neurons supported full differentiation and reproducible assay results, enabling quantitative HTS (qHTS) for cytotoxicity in 1536 well format with a Robust Z’ score of 0.68.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 3","pages":"Article 100143"},"PeriodicalIF":3.1,"publicationDate":"2024-01-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000054/pdfft?md5=6ff5659913d236de92a50d2266c74ac5&pid=1-s2.0-S2472555224000054-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139572293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}