bioRxiv - Biochemistry最新文献_第7页

The isoniazid analog IQG-607 is not a direct substrate for the Mycobacterium tuberculosis catalase-peroxidase KatG 异烟肼类似物 IQG-607 不是结核分枝杆菌过氧化氢酶 KatG 的直接底物

bioRxiv - Biochemistry Pub Date : 2024-09-06 DOI: 10.1101/2024.09.05.611129

Laísa Quadros Barsé, Candida Deves Roth, Adilio da Silva Dadda, Raoní Scheibler Rambo, Pedro Ferrari Dalberto, Kenia Pissinate, José Eduardo Sacconi Nunes, Renata Jardim Etchart, Pablo Machado, Luiz A Basso, Cristiano Valim Bizarro

{"title":"The isoniazid analog IQG-607 is not a direct substrate for the Mycobacterium tuberculosis catalase-peroxidase KatG","authors":"Laísa Quadros Barsé, Candida Deves Roth, Adilio da Silva Dadda, Raoní Scheibler Rambo, Pedro Ferrari Dalberto, Kenia Pissinate, José Eduardo Sacconi Nunes, Renata Jardim Etchart, Pablo Machado, Luiz A Basso, Cristiano Valim Bizarro","doi":"10.1101/2024.09.05.611129","DOIUrl":"https://doi.org/10.1101/2024.09.05.611129","url":null,"abstract":"Tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium tuberculosis (Mtb) and is responsible for millions of deaths. New Mtb strains resistant to TB drugs are emerging and spreading. The first-line TB drug, isoniazid (INH), must be activated inside mycobacterial cells by the catalase-peroxidase enzyme KatG to exert its antimicrobial activity, and mutations on the katG gene are a significant cause of INH resistance in clinics. The metal-containing compound IQG-607 is an INH analog developed to inhibit the target of INH, the FASII enzyme enoyl-ACP-reductase (InhA), without requiring KatG. However, we recently showed that inside mycobacterial cells, IQG-607 activity depends on KatG. Hence, this compound might also be activated by KatG to inhibit InhA. We evaluated whether recombinant MtKatG uses IQG-607 as a substrate in oxidation reactions and adduct formation with NAD+. A recombinant MtKatG was produced in E. coli and purified in a 3-step protocol to obtain a homogeneous protein. An HPLC method was optimized to monitor both oxidation and adduct products, and our assay system was validated by performing control reactions using INH as a substrate. We found that the metal-based compound IQG-607 is not a substrate for recombinant MtKatG under all conditions tested.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"2016 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176085","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Allosteric modulation of the Lon protease by effector binding and local charges 通过效应物结合和局部电荷对 Lon 蛋白酶进行异构调节

bioRxiv - Biochemistry Pub Date : 2024-09-06 DOI: 10.1101/2024.09.06.611642

Justyne L Ogdahl, Peter Chien

{"title":"Allosteric modulation of the Lon protease by effector binding and local charges","authors":"Justyne L Ogdahl, Peter Chien","doi":"10.1101/2024.09.06.611642","DOIUrl":"https://doi.org/10.1101/2024.09.06.611642","url":null,"abstract":"The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon's ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon's DNA binding not only reduces ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon's activity. Based on single molecule measurements we find that this change in activity is correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"63 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Molecular insights into the stimulation of SNM1A nuclease activity by CSB during interstrand crosslink processing 链间交联处理过程中 CSB 刺激 SNM1A 核酸酶活性的分子见解

bioRxiv - Biochemistry Pub Date : 2024-09-06 DOI: 10.1101/2024.09.05.611390

Rebecca Roddan, William J. Nathan, Joseph A. Newman, Afaf H. El-Sagheer, David M. Wilson, Tom Brown, Christopher J. Schofield, Peter J. McHugh

{"title":"Molecular insights into the stimulation of SNM1A nuclease activity by CSB during interstrand crosslink processing","authors":"Rebecca Roddan, William J. Nathan, Joseph A. Newman, Afaf H. El-Sagheer, David M. Wilson, Tom Brown, Christopher J. Schofield, Peter J. McHugh","doi":"10.1101/2024.09.05.611390","DOIUrl":"https://doi.org/10.1101/2024.09.05.611390","url":null,"abstract":"The SNM1A exonuclease plays a key role in repair of interstrand crosslinks (ICLs) which represent a particularly toxic class of DNA damage. Previous work suggests that the SWI/SNF family ATP-dependent, chromatin remodeler, Cockayne Syndrome B protein (CSB) interacts with SNM1A, during transcription-coupled DNA interstrand crosslink repair (TC-ICL repair). Here, we validate this interaction using purified proteins and demonstrate that the ubiquitin-binding and winged-helix domains of CSB are required for interaction with the catalytic domain of SNM1A. The winged helix domain is essential for binding, although high-affinity SNM1A binding requires the entire CSB C-terminal region (residues 1187-1493), where two copies of the C-terminal domain of CSB are necessary for a stable interaction with SNM1A. CSB stimulates SNM1A nuclease activity on varied model DNA repair intermediate substrates. Importantly, CSB was observed to stimulate digestion through ICLs in vitro, implying a key role of the interaction in ′unhooking′ during TC-ICL repair. AlphaFold3 models of CSB constructs complexed with the SNM1A catalytic domain enabled mapping of the molecular contacts required for the CSB-SNM1A interaction. This identified specific protein-protein interactions necessary for CSB′s stimulation of SNM1A′s activity that we confirmed experimentally. Additionally, our studies reveal the C-terminal region of CSB as a novel DNA binding region that also is involved in stimulation of SNM1A-mediated ICL repair. Moreover, targeting protein-protein interactions that are vital for specific nuclease activities, such as CSB′s stimulation of SNM1A′s nuclease activity, may be a productive alternative therapeutic strategy to nuclease active site inhibition.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176063","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Next generation APOBEC3 inhibitors: Optimally designed for potency and nuclease stability 新一代 APOBEC3 抑制剂：针对效力和核酸酶稳定性的优化设计

bioRxiv - Biochemistry Pub Date : 2024-09-06 DOI: 10.1101/2024.09.05.611238

Adam K Hedger, Wazo Myint, Jeong Min Lee, Diego Suchenski-Loustaunau, Vanivilasini Balachandran, Ala M Shaqra, Nese Kurt Yilmaz, Jonathan Watts, Hiroshi Matsuo, Celia A Schiffer

{"title":"Next generation APOBEC3 inhibitors: Optimally designed for potency and nuclease stability","authors":"Adam K Hedger, Wazo Myint, Jeong Min Lee, Diego Suchenski-Loustaunau, Vanivilasini Balachandran, Ala M Shaqra, Nese Kurt Yilmaz, Jonathan Watts, Hiroshi Matsuo, Celia A Schiffer","doi":"10.1101/2024.09.05.611238","DOIUrl":"https://doi.org/10.1101/2024.09.05.611238","url":null,"abstract":"APOBEC3 (or A3) enzymes have emerged as potential therapeutic targets due to their role in introducing heterogeneity in viruses and cancer, often leading to drug resistance. Inhibiting these enzymes has remained elusive as initial phosphodiester (PO) linked DNA based inhibitors lack stability and potency. We have enhanced both potency and nuclease stability, of 2'-deoxy-zebularine (dZ), substrate-based oligonucleotide inhibitors for two critical A3s: A3A and A3G. While replacing the phosphate backbone with phosphorothioate (PS) linkages increased nuclease stability, fully PS-modified inhibitors lost potency (1.4-3.7 fold) due to the structural constraints of the active site. For both enzymes, mixed PO/PS backbones enhanced potency (2.3-9.2 fold), while also vastly improving nuclease resistance. We also strategically introduced 2'-fluoro sugar modifications, creating the first nanomolar inhibitor of A3G-CTD2. With hairpin-structured inhibitors containing optimized PS patterns and LNA sugar modifications, we characterize the first single-digit nanomolar inhibitor targeting A3A. These extremely potent A3A inhibitors, were highly resistant to nuclease degradation in serum stability assays. Overall, our optimally designed A3 oligonucleotide inhibitors show improved potency and stability, compared to previous attempts to inhibit these critical enzymes, opening the door to realize the therapeutic potential of A3 inhibition.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

USP24 is an ISG15 cross-reactive deubiquitinase that mediates IFN-I production by de-ISGylating the RNA helicase MOV10 USP24 是一种 ISG15 交叉反应型去泛素化酶，它通过去 ISGyl 化 RNA 螺旋酶 MOV10 来介导 IFN-I 的产生。

bioRxiv - Biochemistry Pub Date : 2024-09-06 DOI: 10.1101/2024.09.06.611391

Rishov Mukhopadhyay, Simeon D Draganov, Jimmy J.L.L Akkermans, Marjolein Kikkert, Klaus-Peter Knobeloch, Günter Fritz, María Guzmán, Sonia Zuñiga, Robbert Q Kim, Benedikt M Kessler, Adán Pinto-Fernández, Paul P Geurink, Aysegul Sapmaz

{"title":"USP24 is an ISG15 cross-reactive deubiquitinase that mediates IFN-I production by de-ISGylating the RNA helicase MOV10","authors":"Rishov Mukhopadhyay, Simeon D Draganov, Jimmy J.L.L Akkermans, Marjolein Kikkert, Klaus-Peter Knobeloch, Günter Fritz, María Guzmán, Sonia Zuñiga, Robbert Q Kim, Benedikt M Kessler, Adán Pinto-Fernández, Paul P Geurink, Aysegul Sapmaz","doi":"10.1101/2024.09.06.611391","DOIUrl":"https://doi.org/10.1101/2024.09.06.611391","url":null,"abstract":"The interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I Interferon (IFN-I) and plays a crucial role in the innate immune response against viral infections. ISG15 is conjugated to target proteins by an enzymatic cascade through a process called ISGylation. While ubiquitin-specific protease 18 (USP18) is a well-defined deISGylase counteracting ISG15 conjugation, ISG15 cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Our study reports USP24 as a novel ISG15 cross-reactive DUB identified through activity-based protein profiling (ABPP). We demonstrate that recombinant USP24 processed pro-ISG15 and ISG15-linked synthetic substrates in vitro. Moreover, the depletion of USP24 significantly increased the accumulation of ISG15 conjugates upon IFN-β stimulation. An extensive proteomic analysis of the USP24-dependent ISGylome, integrating total proteome, GG-peptidome, and ISG15 interactome data, identified the helicase Moloney leukemia virus 10 (MOV10) as a specific target of USP24 for deISGylation. Further validation in cells revealed that ISGylated MOV10 enhances IFN-β production/secretion, whereas USP24 deISGylates MOV10 to negatively regulate the innate immune response. This study showcases USP24's novel roles in modulating ISGylation and modulation of the IFN-I-dependent immune responses, with potential therapeutic implications in infectious diseases, cancer, autoimmunity, and neuroinflammation.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase 对双特异性磷酸酶 MKP5 异构调控的动态和结构见解

bioRxiv - Biochemistry Pub Date : 2024-09-05 DOI: 10.1101/2024.09.05.611520

Erin Skeens, Federica Maschietto, Manjula Ramu, Shanelle Shillingford, Elias J Lolis, Victor S Batista, Anton M Bennett, George P Lisi

{"title":"Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase","authors":"Erin Skeens, Federica Maschietto, Manjula Ramu, Shanelle Shillingford, Elias J Lolis, Victor S Batista, Anton M Bennett, George P Lisi","doi":"10.1101/2024.09.05.611520","DOIUrl":"https://doi.org/10.1101/2024.09.05.611520","url":null,"abstract":"Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

CRISPR-Cas9 target-strand nicking provides phage resistance by inhibiting replication 通过抑制复制，CRISPR-Cas9 靶向链切分技术提供了抗噬菌体能力

bioRxiv - Biochemistry Pub Date : 2024-09-05 DOI: 10.1101/2024.09.05.611540

Giang T Nguyen, Michael A Schelling, Dipali G Sashital

{"title":"CRISPR-Cas9 target-strand nicking provides phage resistance by inhibiting replication","authors":"Giang T Nguyen, Michael A Schelling, Dipali G Sashital","doi":"10.1101/2024.09.05.611540","DOIUrl":"https://doi.org/10.1101/2024.09.05.611540","url":null,"abstract":"Cas endonucleases, like Cas9 and Cas12a, are RNA-guided immune effectors that provide bacterial defense against bacteriophages. Cas endonucleases rely on divalent metal ions for their enzymatic activities and to facilitate conformational changes that are required for specific recognition and cleavage of target DNA. While Cas endonucleases typically produce double-strand breaks (DSBs) in DNA targets, reduced, physiologically relevant Mg2+ concentrations and target mismatches can result in incomplete second-strand cleavage, resulting in the production of a nicked DNA. It remains poorly understood whether nicking by Cas endonucleases is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only one of two nuclease domains is catalytically active. By testing a large panel of guide RNAs, we find that target strand nicking can be sufficient to provide immunity, while non-target nicking does not provide any additional protection beyond Cas9 binding. Target-strand nicking inhibits phage replication and can reduce the susceptibility of Cas9 to viral escape when targeting non-essential regions of the genome. Cleavage of the non-target strand by the RuvC domain is strongly impaired at low Mg2+ concentrations. As a result, fluctuations in the concentration of other biomolecules that can compete for binding of free Mg2+ strongly influences the ability of Cas9 to form a DSB at targeted sites. Overall, our results suggest that Cas9 may only nick DNA during CRISPR-mediated immunity, especially under conditions of low Mg2+ availability in cells.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"116 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The Main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII 来自 SARS-CoV-2 的主蛋白酶（Mpro）通过激活凝血因子 VII 和 FXII 触发体外血浆凝结

bioRxiv - Biochemistry Pub Date : 2024-09-05 DOI: 10.1101/2024.09.05.611400

Anna Pagotto, Federico Uliana, Giulia Nordio, Andrea Pietrangelini, Laura Acquasaliente, Maria Ludovica Macchia, Massimo Bellanda, Barbara Gatto, Giustina De Silvestro, Piero Marson, Paolo Simioni, Paola Picotti, Vincenzo De Filippis

{"title":"The Main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII","authors":"Anna Pagotto, Federico Uliana, Giulia Nordio, Andrea Pietrangelini, Laura Acquasaliente, Maria Ludovica Macchia, Massimo Bellanda, Barbara Gatto, Giustina De Silvestro, Piero Marson, Paolo Simioni, Paola Picotti, Vincenzo De Filippis","doi":"10.1101/2024.09.05.611400","DOIUrl":"https://doi.org/10.1101/2024.09.05.611400","url":null,"abstract":"Although the connection between COVID-19 and coagulopathy has been clear since the early days of SARS-CoV-2 pandemic, the underlying molecular mechanisms remain unclear. Available data support that the burst of cytokines and bradykinin, observed in some COVID-19 patients, sustains systemic inflammation and the hypercoagulant state, thus increasing thrombotic risk. Here we show that the SARS-CoV-2 main protease (Mpro) can play a direct role in the activation of the coagulation cascade. Adding Mpro to human plasma from healthy donors increased clotting probability by 2.5-fold. The results of enzymatic assays and degradomics analysis indicate that Mpro triggers plasma clotting by proteolytically activating coagulation factors zymogens VII and XII at their physiological activation sites, i.e. Arg152-Ile153 bond for FVII and Arg353-Val354 bond for FXII, where FVII and FXII are strategically positioned at the very beginning of the extrinsic or intrinsic pathways of blood coagulation. These findings are not compatible with the substrate specificity of the protease known so far, displaying a prevalence for a Gln-residue in P1 and a hydrophobic amino acid in P2 position. This apparent discrepancy was resolved by High Throughput Protease Screen assay, unveiling an extended, time-dependent, secondary specificity of Mpro for Arg-X bonds, which was further confirmed by Hydrogen-Deuterium Exchange Mass spectrometry analysis of Arg-containing inhibitors binding to Mpro and by enzymatic assays showing that the protease can cleave peptide substrates containing Arg in P1. Overall, integrating biochemical, proteomics and structural biology experiments, we unveil a novel mechanism linking SARS-CoV-2 infection to thrombotic complications in COVID-19.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

STRUCTURE-BASED DESIGN OF PROTACS FOR THE DEGRADATION OF SOLUBLE EPOXIDE HYDROLASE 以结构为基础设计降解可溶性环氧化物水解酶的原药

bioRxiv - Biochemistry Pub Date : 2024-09-05 DOI: 10.1101/2024.09.05.611393

Julia Schoenfeld, Steffen Brunst, Ludmila Ciomirtan, Nick Liebisch, Adarsh Kumar, Johanna Ehrler, Lukas Wintermeier, Jan Heering, Astrid Brueggerhof, Lilia Weizel, Astrid Kahnt, Manfred Schubert-Zsilavecz, Stefan Knapp, Robert Fuerst, Eugen Proschak, Kerstin Hiesinger

{"title":"STRUCTURE-BASED DESIGN OF PROTACS FOR THE DEGRADATION OF SOLUBLE EPOXIDE HYDROLASE","authors":"Julia Schoenfeld, Steffen Brunst, Ludmila Ciomirtan, Nick Liebisch, Adarsh Kumar, Johanna Ehrler, Lukas Wintermeier, Jan Heering, Astrid Brueggerhof, Lilia Weizel, Astrid Kahnt, Manfred Schubert-Zsilavecz, Stefan Knapp, Robert Fuerst, Eugen Proschak, Kerstin Hiesinger","doi":"10.1101/2024.09.05.611393","DOIUrl":"https://doi.org/10.1101/2024.09.05.611393","url":null,"abstract":"Soluble epoxide hydrolase (sEH) represents a promising target for inflammation-related diseases as it hydrolyzes highly anti-inflammatory epoxy-fatty acids (EpFAs) to the less active corresponding diols.1 sEH harbours two distinct catalytic domains, the C-terminal hydrolase domain and the N-terminal phosphatase domain which are connected by a proline-rich linker. Although potent inhibitors of enzymatic activity are available for both domains, sEH-PROTACs offer the unique ability to simultaneously degrade both domains, mimicking the sEH knockout phenotype associated with beneficial effects as reducing inflammation, attenuating neuroinflammation, and delaying the progression of Alzheimer's disease. Herein, we report the structure-based development of a potent sEH-PROTAC as a useful tool compound for the investigation of sEH. In order to facilitate a rapid testing of the synthesized compounds a cell-based sEH degradation assay was developed based on the HiBiT-technology. A structure-activity-relationship (SAR) investigation was performed, based on the crystal structure of previously published sEH inhibitor FL217 where we identified two possible exit vectors. We designed and synthesized a set of 24 PROTACs with varying linkers in a combinatorial manner. Furthermore, co-crystallization of sEH with two selected PROTACs allowed us to explore the binding mode and rationalize the appropriate linker length. After biological and physicochemical investigation, the most suitable PROTAC 23 was identified and applied to degrade sEH in primary human macrophages, marking the successful translation and applicability to non-artificial systems.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"75 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone 基于人工智能的 KATP 通道药物配体的发现和低温电子显微镜结构阐释

bioRxiv - Biochemistry Pub Date : 2024-09-05 DOI: 10.1101/2024.09.05.611490

Assmaa Elsheikh, Camden M Driggers, Ha H. Truong, Zhongying Yang, John Allen, Niel Henriksen, Katarzyna Walczewska-Szewc, Show-Ling Shyng

{"title":"AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone","authors":"Assmaa Elsheikh, Camden M Driggers, Ha H. Truong, Zhongying Yang, John Allen, Niel Henriksen, Katarzyna Walczewska-Szewc, Show-Ling Shyng","doi":"10.1101/2024.09.05.611490","DOIUrl":"https://doi.org/10.1101/2024.09.05.611490","url":null,"abstract":"Pancreatic KATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the KATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used KATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for KATP trafficking impaired CHI is hindered by high-affinity binding, which limits functional recovery of rescued channels. Recent structural studies of KATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known KATP pharmacochaperones bind. The structural knowledge provides a framework for discovering KATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on KATP channel trafficking mutations. Aekatperone reversibly inhibits KATP channel activity with a half-maximal inhibitory concentration (IC50) ~ 9 μM. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of KATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a KATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by KATP trafficking defects.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"60 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142223285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0