JACS Au最新文献

{"title":"Non-Markovian Dynamic Models Identify Non-Canonical KRAS-VHL Encounter Complex Conformations for Novel PROTAC Design","authors":"Yunrui Qiu,&nbsp;Rafal P. Wiewiora,&nbsp;Jesus A. Izaguirre,&nbsp;Huafeng Xu,&nbsp;Woody Sherman,&nbsp;Weiping Tang* and Xuhui Huang*,&nbsp;","doi":"10.1021/jacsau.4c0050310.1021/jacsau.4c00503","DOIUrl":"https://doi.org/10.1021/jacsau.4c00503https://doi.org/10.1021/jacsau.4c00503","url":null,"abstract":"<p >Targeted protein degradation (TPD) is emerging as a promising therapeutic approach for cancer and other diseases, with an increasing number of programs demonstrating its efficacy in human clinical trials. One notable method for TPD is Proteolysis Targeting Chimeras (PROTACs) that selectively degrade a protein of interest (POI) through E3-ligase induced ubiquitination followed by proteasomal degradation. PROTACs utilize a warhead-linker-ligand architecture to bring the POI (bound to the warhead) and the E3 ligase (bound to the ligand) into proximity. The resulting non-native protein–protein interactions (PPIs) formed between the POI and E3 ligase lead to the formation of a stable ternary complex, enhancing cooperativity for TPD. A significant challenge in PROTAC design is the screening of the linkers to induce favorable non-native PPIs between POI and E3 ligase. Here, we present a physics-based computational protocol to predict noncanonical and metastable PPI interfaces between an E3 ligase and a given POI, aiding in the design of linkers to stabilize the ternary complex and enhance degradation. Specifically, we build the non-Markovian dynamic model using the Integrative Generalized Master equation (IGME) method from ∼1.5 ms all-atom molecular dynamics simulations of linker-less encounter complex, to systematically explore the inherent PPIs between the oncogene homologue protein and the von Hippel-Lindau E3 ligase. Our protocol revealed six metastable states each containing a different PPI interface. We selected three of these metastable states containing promising PPIs for linker design. Our selection criterion included thermodynamic and kinetic stabilities of PPIs and the accessibility between the solvent-exposed sites on the warheads and E3 ligand. One selected PPIs closely matches a recent cocrystal PPI interface structure induced by an experimentally designed PROTAC with potent degradation efficacy. We anticipate that our protocol has significant potential for widespread application in predicting metastable POI-ligase interfaces that can enable rational design of PROTACs.</p>","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jacsau.4c00503","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142550421","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Short Repeat Ribonucleic Acid Reduces Cytotoxicity by Preventing the Aggregation of TDP-43 and Its 25 KDa Carboxy-Terminal Fragment.","authors":"Ai Fujimoto, Masataka Kinjo, Akira Kitamura","doi":"10.1021/jacsau.4c00566","DOIUrl":"10.1021/jacsau.4c00566","url":null,"abstract":"<p><p>TAR DNA/RNA-binding protein 43 kDa (TDP-43) proteinopathy is a hallmark of neurodegenerative disorders, such as amyotrophic lateral sclerosis, in which cytoplasmic aggregates containing TDP-43 and its C-terminal fragments, such as TDP-25, are observed in degenerative neuronal cells. However, few reports have focused on small molecules that can reduce their aggregation and cytotoxicity. Here, we show that short RNA repeats of GGGGCC and AAAAUU are aggregation suppressors of TDP-43 and TDP-25. TDP-25 interacts with these RNAs, as well as TDP-43, despite the lack of major RNA-recognition motifs using fluorescence cross-correlation spectroscopy. Expression of these RNAs significantly decreases the number of cells harboring cytoplasmic aggregates of TDP-43 and TDP-25 and ameliorates cell death by TDP-25 and mislocalized TDP-43 without altering the cellular transcriptome of molecular chaperones. Consequently, short RNA repeats of GGGGCC and AAAAUU can maintain proteostasis by preventing the aggregation of TDP-43 and TDP-25.</p>","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522920/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142559873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Non-Markovian Dynamic Models Identify Non-Canonical KRAS-VHL Encounter Complex Conformations for Novel PROTAC Design.","authors":"Yunrui Qiu, Rafal P Wiewiora, Jesus A Izaguirre, Huafeng Xu, Woody Sherman, Weiping Tang, Xuhui Huang","doi":"10.1021/jacsau.4c00503","DOIUrl":"10.1021/jacsau.4c00503","url":null,"abstract":"<p><p>Targeted protein degradation (TPD) is emerging as a promising therapeutic approach for cancer and other diseases, with an increasing number of programs demonstrating its efficacy in human clinical trials. One notable method for TPD is Proteolysis Targeting Chimeras (PROTACs) that selectively degrade a protein of interest (POI) through E3-ligase induced ubiquitination followed by proteasomal degradation. PROTACs utilize a warhead-linker-ligand architecture to bring the POI (bound to the warhead) and the E3 ligase (bound to the ligand) into proximity. The resulting non-native protein-protein interactions (PPIs) formed between the POI and E3 ligase lead to the formation of a stable ternary complex, enhancing cooperativity for TPD. A significant challenge in PROTAC design is the screening of the linkers to induce favorable non-native PPIs between POI and E3 ligase. Here, we present a physics-based computational protocol to predict noncanonical and metastable PPI interfaces between an E3 ligase and a given POI, aiding in the design of linkers to stabilize the ternary complex and enhance degradation. Specifically, we build the non-Markovian dynamic model using the Integrative Generalized Master equation (IGME) method from ∼1.5 ms all-atom molecular dynamics simulations of linker-less encounter complex, to systematically explore the inherent PPIs between the oncogene homologue protein and the von Hippel-Lindau E3 ligase. Our protocol revealed six metastable states each containing a different PPI interface. We selected three of these metastable states containing promising PPIs for linker design. Our selection criterion included thermodynamic and kinetic stabilities of PPIs and the accessibility between the solvent-exposed sites on the warheads and E3 ligand. One selected PPIs closely matches a recent cocrystal PPI interface structure induced by an experimentally designed PROTAC with potent degradation efficacy. We anticipate that our protocol has significant potential for widespread application in predicting metastable POI-ligase interfaces that can enable rational design of PROTACs.</p>","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522902/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142559869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In-Cell DEER Spectroscopy of Nanodisc-Delivered Membrane Proteins in Living Cell Membranes","authors":"Chu-Chun Cheng,&nbsp;Ruei-Fong Tsai,&nbsp;Che-Kai Lin,&nbsp;Kui-Thong Tan,&nbsp;Vidmantas Kalendra,&nbsp;Mantas Simenas,&nbsp;Chun-Wei Lin* and Yun-Wei Chiang*,&nbsp;","doi":"10.1021/jacsau.4c0070210.1021/jacsau.4c00702","DOIUrl":"https://doi.org/10.1021/jacsau.4c00702https://doi.org/10.1021/jacsau.4c00702","url":null,"abstract":"<p >Membrane proteins are integral to numerous cellular processes, yet their conformational dynamics in native environments remains difficult to study. This study introduces a nanodelivery method using nanodiscs to transport spin-labeled membrane proteins into the membranes of living cells, enabling direct in-cell double electron–electron resonance (DEER) spectroscopy measurements. We investigated the membrane protein BsYetJ, incorporating spin labels at key positions to monitor conformational changes. Our findings demonstrate successful delivery and high-quality DEER data for BsYetJ in both Gram-negative <i>E. coli</i> and Gram-positive <i>B. subtilis</i> membranes. The delivered BsYetJ retains its ability to transport calcium ions. DEER analysis reveals distinct conformational states of BsYetJ in different membrane environments, highlighting the influence of lipid composition on the protein structure. This nanodelivery method overcomes traditional limitations, enabling the study of membrane proteins in more physiologically relevant conditions.</p>","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jacsau.4c00702","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142517691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In-Cell DEER Spectroscopy of Nanodisc-Delivered Membrane Proteins in Living Cell Membranes.","authors":"Chu-Chun Cheng, Ruei-Fong Tsai, Che-Kai Lin, Kui-Thong Tan, Vidmantas Kalendra, Mantas Simenas, Chun-Wei Lin, Yun-Wei Chiang","doi":"10.1021/jacsau.4c00702","DOIUrl":"10.1021/jacsau.4c00702","url":null,"abstract":"<p><p>Membrane proteins are integral to numerous cellular processes, yet their conformational dynamics in native environments remains difficult to study. This study introduces a nanodelivery method using nanodiscs to transport spin-labeled membrane proteins into the membranes of living cells, enabling direct in-cell double electron-electron resonance (DEER) spectroscopy measurements. We investigated the membrane protein BsYetJ, incorporating spin labels at key positions to monitor conformational changes. Our findings demonstrate successful delivery and high-quality DEER data for BsYetJ in both Gram-negative <i>E. coli</i> and Gram-positive <i>B. subtilis</i> membranes. The delivered BsYetJ retains its ability to transport calcium ions. DEER analysis reveals distinct conformational states of BsYetJ in different membrane environments, highlighting the influence of lipid composition on the protein structure. This nanodelivery method overcomes traditional limitations, enabling the study of membrane proteins in more physiologically relevant conditions.</p>","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522923/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142559868","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"2,2-Difluoro Derivatives of Fucose Can Inhibit Cell Surface Fucosylation without Causing Slow Transfer to Acceptors.","authors":"Yanyan Liu, Igor R Sweet, Geert-Jan Boons","doi":"10.1021/jacsau.4c00681","DOIUrl":"10.1021/jacsau.4c00681","url":null,"abstract":"<p><p>Fucosyl transferases (FUTs) are enzymes that transfer fucose (Fuc) from GDP-Fuc to acceptor substrates, resulting in fucosylated glycoconjugates that are involved in myriad physiological and disease processes. Previously, it has been shown that per-<i>O</i>-acetylated 2-F-Fuc can be taken up by cells and converted into GDP-2-F-Fuc, which is a competitive inhibitor of FUTs. Furthermore, it can act as a feedback inhibitor of <i>de novo</i> biosynthesis of GDP-Fuc resulting in reduced glycoconjugate fucosylation. However, GDP-2-F-Fuc and several other reported analogues are slow substrates, which can result in unintended incorporation of unnatural fucosides. Here, we describe the design, synthesis, and biological evaluation of GDP-2,2-di-F-Fuc and the corresponding prodrugs as an inhibitor of FUTs. This compound lacks the slow transfer activity observed for the monofluorinated counterpart. Furthermore, it was found that GDP-2-F-Fuc and GDP-2,2-di-F-Fuc have similar <i>K_i</i> values for the various human fucosyl transferases, while the corresponding phosphate prodrugs exhibit substantial differences in inhibition of cell surface fucosylation. Quantitative sugar nucleotide analysis by Liquid chromatography-mass spectrometry (LC-MS) indicates that the 2,2-di-F-Fuc prodrug has substantially greater feedback inhibitory activity. It was also found that by controlling the concentration of the inhibitor, varying degrees of inhibition of the biosynthesis of different types of fucosylated <i>N-</i>glycan structures can be achieved. These findings open new avenues for the modulation of fucosylation of cell surface glycoconjugates.</p>","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522930/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142559862","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural Plasticity within 3-Hydroxy-3-Methylglutaryl Synthases Catalyzing the First Step of β-Branching in Polyketide Biosynthesis Underpins a Dynamic Mechanism of Substrate Accommodation","authors":"Sabrina Collin*,&nbsp;Kira J. Weissman* and Arnaud Gruez*,&nbsp;","doi":"10.1021/jacsau.4c0047710.1021/jacsau.4c00477","DOIUrl":"https://doi.org/10.1021/jacsau.4c00477https://doi.org/10.1021/jacsau.4c00477","url":null,"abstract":"<p >Understanding how enzymes have been repurposed by evolution to carry out new functions is a key goal of mechanistic enzymology. In this study we aimed to identify the adaptations required to allow the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) enzymes of primary isoprenoid assembly to function in specialized polyketide biosynthetic pathways, where they initiate β-branching. This role notably necessitates that the HMG synthases (HMGSs) act on substrates tethered to noncatalytic acyl carrier protein (ACP) domains instead of coenzyme A, and accommodation of substantially larger chains within the active sites. Here, we show using a combination of X-ray crystallography and small-angle X-ray scattering, that a model HMGS from the virginiamycin system exhibits markedly increased flexibility relative to its characterized HMGCS counterparts. This mobility encompasses multiple secondary structural elements that define the dimensions and chemical nature of the active site, as well the catalytic residues themselves. This result was unexpected given the well-ordered character of the HMGS within the context of an HMGS/ACP complex, but analysis by synchrotron radiation circular dichroism demonstrates that this interaction leads to increased HMGS folding. This flexible to more rigid transition is notably not accounted for by AlphaFold2, which yielded a structural model incompatible with binding of the native substrates. Taken together, these results illustrate the continued necessity of an integrative structural biology approach combining crystallographic and solution-phase data for elucidating the mechanisms underlying enzyme remodeling, information which can inform strategies to replicate such evolution effectively in the laboratory.</p>","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jacsau.4c00477","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142517732","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Frontiers in Solar Photovoltaic Materials.","authors":"Justin M Hodgkiss","doi":"10.1021/jacsau.4c00771","DOIUrl":"https://doi.org/10.1021/jacsau.4c00771","url":null,"abstract":"","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11423318/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142335228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural Plasticity within 3-Hydroxy-3-Methylglutaryl Synthases Catalyzing the First Step of β-Branching in Polyketide Biosynthesis Underpins a Dynamic Mechanism of Substrate Accommodation.","authors":"Sabrina Collin, Kira J Weissman, Arnaud Gruez","doi":"10.1021/jacsau.4c00477","DOIUrl":"10.1021/jacsau.4c00477","url":null,"abstract":"<p><p>Understanding how enzymes have been repurposed by evolution to carry out new functions is a key goal of mechanistic enzymology. In this study we aimed to identify the adaptations required to allow the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) enzymes of primary isoprenoid assembly to function in specialized polyketide biosynthetic pathways, where they initiate β-branching. This role notably necessitates that the HMG synthases (HMGSs) act on substrates tethered to noncatalytic acyl carrier protein (ACP) domains instead of coenzyme A, and accommodation of substantially larger chains within the active sites. Here, we show using a combination of X-ray crystallography and small-angle X-ray scattering, that a model HMGS from the virginiamycin system exhibits markedly increased flexibility relative to its characterized HMGCS counterparts. This mobility encompasses multiple secondary structural elements that define the dimensions and chemical nature of the active site, as well the catalytic residues themselves. This result was unexpected given the well-ordered character of the HMGS within the context of an HMGS/ACP complex, but analysis by synchrotron radiation circular dichroism demonstrates that this interaction leads to increased HMGS folding. This flexible to more rigid transition is notably not accounted for by AlphaFold2, which yielded a structural model incompatible with binding of the native substrates. Taken together, these results illustrate the continued necessity of an integrative structural biology approach combining crystallographic and solution-phase data for elucidating the mechanisms underlying enzyme remodeling, information which can inform strategies to replicate such evolution effectively in the laboratory.</p>","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522927/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142559876","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"2,2-Difluoro Derivatives of Fucose Can Inhibit Cell Surface Fucosylation without Causing Slow Transfer to Acceptors","authors":"Yanyan Liu,&nbsp;Igor R. Sweet and Geert-Jan Boons*,&nbsp;","doi":"10.1021/jacsau.4c0068110.1021/jacsau.4c00681","DOIUrl":"https://doi.org/10.1021/jacsau.4c00681https://doi.org/10.1021/jacsau.4c00681","url":null,"abstract":"<p >Fucosyl transferases (FUTs) are enzymes that transfer fucose (Fuc) from GDP-Fuc to acceptor substrates, resulting in fucosylated glycoconjugates that are involved in myriad physiological and disease processes. Previously, it has been shown that per-<i>O</i>-acetylated 2-F-Fuc can be taken up by cells and converted into GDP-2-F-Fuc, which is a competitive inhibitor of FUTs. Furthermore, it can act as a feedback inhibitor of <i>de novo</i> biosynthesis of GDP-Fuc resulting in reduced glycoconjugate fucosylation. However, GDP-2-F-Fuc and several other reported analogues are slow substrates, which can result in unintended incorporation of unnatural fucosides. Here, we describe the design, synthesis, and biological evaluation of GDP-2,2-di-F-Fuc and the corresponding prodrugs as an inhibitor of FUTs. This compound lacks the slow transfer activity observed for the monofluorinated counterpart. Furthermore, it was found that GDP-2-F-Fuc and GDP-2,2-di-F-Fuc have similar <i>K_i</i> values for the various human fucosyl transferases, while the corresponding phosphate prodrugs exhibit substantial differences in inhibition of cell surface fucosylation. Quantitative sugar nucleotide analysis by Liquid chromatography–mass spectrometry (LC–MS) indicates that the 2,2-di-F-Fuc prodrug has substantially greater feedback inhibitory activity. It was also found that by controlling the concentration of the inhibitor, varying degrees of inhibition of the biosynthesis of different types of fucosylated <i>N-</i>glycan structures can be achieved. These findings open new avenues for the modulation of fucosylation of cell surface glycoconjugates.</p>","PeriodicalId":94060,"journal":{"name":"JACS Au","volume":null,"pages":null},"PeriodicalIF":8.5,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jacsau.4c00681","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142550528","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}