ACS Bio & Med Chem Au最新文献

{"title":"Shedding Light on the Elephant in a Dark Room in the Discovery of New Medicine: Highlighting Molecular Pharmaceutics within ACS Bio & Med Chem Au","authors":"Afsaneh Lavasanifar*,  and , Lynne S. Taylor, ","doi":"10.1021/acsbiomedchemau.2c00047","DOIUrl":"https://doi.org/10.1021/acsbiomedchemau.2c00047","url":null,"abstract":"T parable of the “Blind men and an elephant” is a famous story in many cultures around the world. The story, which originated in the ancient Indian subcontinent, was retold by Rumi, the 13th century Persian poet, in a poem named “The elephant in the dark room”. In the 19th century, the American poet John Godfrey Saxe made his own poem based on the story (The poems of John Godfrey Saxe/The Blind Men and the Elephant). The story tells us about six blind men, who have not seen an elephant before, approaching one in a dark room. They try to learn what the elephant is like by touching it. Each man feels a different part of the elephant’s body (its side, tusk, trunk etc.) and describes it based on their experience, which of course is different from the description of others and far from reality or the big picture. This tale is a great metaphor for the limitations of isolated scientific observation in the development of real and accurate knowledge and/or the translation of discoveries into impactful solutions for real-life problems. Reflecting on the lessons learned from this tale, as applied to the scientific world, reminds us of the need for a multidisciplinary and collaborative approach for producing impactful research, and this is particularly true for the drug development process. A medicine is much more than a molecule and its development requires contributions from different scientific disciplines including, but not limited to, medicinal and analytical chemists, biologists, and formulation scientists. The key to life-changing new discoveries in drug development is in the communication and exchange of ideas between scientific teams from these different disciplines. Recognizing the need for a fully open access, multidisciplinary scientific communication platform, particularly for scientists working in drug development, has led the American Chemical Society to launch ACS Bio & Med Chem Au. The journal, which is one of the nine gold (Au) open access journals, has a broad scope and showcases research from biological, medicinal, and pharmaceutical sciences to nurture communication and information sharing between scientists from different but related disciplines, and this is hugely useful for drug development research. ACS Bio & Med Chem Au particularly recognizes the importance of the molecular and mechanistic understanding of drug formulations as well as translational research in areas of pharmaceutical chemistry, existing and emerging drug delivery systems, biological performance of formulations, and other multidisciplinary research projects, especially those in the field of pharmaceutical development which are within the scope of Molecular Pharmaceutics. In this context, the journal welcomes submission of Articles, Letters, Perspectives, and Reviews with a focus on understanding the physicochemical properties of drugs and drug formulations (including small molecules, proteins, and genes) affecting their in vitro/in vivo function, the development of ","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 4","pages":"313–315"},"PeriodicalIF":0.0,"publicationDate":"2022-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.2c00047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72198245","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":"Modeling the Effect of Cooperativity in Ternary Complex Formation and Targeted Protein Degradation Mediated by Heterobifunctional Degraders","authors":"D. Park, J. Izaguirre, R. Coffey, Hu Xu","doi":"10.1101/2022.03.22.485399","DOIUrl":"https://doi.org/10.1101/2022.03.22.485399","url":null,"abstract":"Chemically induced proximity between certain endogenous enzymes and a protein of interest (POI) inside cells may cause post-translational modifications to the POI with biological consequences and potential therapeutic effects. Heterobifunctional (HBF) molecules that bind with one functional part to a target POI and with the other to an E3 ligase induce the formation of a target-HBF-E3 ternary complex, which can lead to ubiquitination and proteasomal degradation of the POI. Targeted protein degra-dation (TPD) by HBFs offers a promising approach to modulating disease-associated proteins, especially those that are intractable using other therapeutic approaches, such as enzymatic inhibition. The three-way interactions among the HBF, the target POI, and the ligase—including the protein-protein interaction (PPI) between the POI and the ligase—contribute to the stability of the ternary complex, manifested as positive or negative binding cooperativity in its formation. How such cooperativity affects HBF-mediated degradation is an open question. In this work, we develop a pharmaco-dynamic model that describes the kinetics of the key reactions in the TPD process, and we use this model to investigate the role of cooperativity in the ternary complex formation and in the target POI degradation. Our model predicts that, under certain conditions, increasing cooperativity may diminish degradation, implying an optimal range of cooperativity values for efficient degradation. We also develop a statistical inference model for determining cooperativity in intracellular ternary complex formation from cellular assay data, and demonstrate it by quantifying the change in cooperativity due to site-directed mutagenesis at the POI-ligase interface of the SMARCA2-ACBI1-VHL ternary complex. Our pharmacodynamic model provides a quantitative framework to dissect the complex HBF-mediated TPD process and may inform the rational design of effective HBF degraders.","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"3 1","pages":"74 - 86"},"PeriodicalIF":0.0,"publicationDate":"2022-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46056866","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}

{"title":"Plant Cysteine Oxidase Oxygen-Sensing Function Is Conserved in Early Land Plants and Algae","authors":"Leah J. Taylor-Kearney,&nbsp;Samuel Madden,&nbsp;Jack Wilson,&nbsp;William K. Myers,&nbsp;Dona M. Gunawardana,&nbsp;Elisabete Pires,&nbsp;Philip Holdship,&nbsp;Anthony Tumber,&nbsp;Rosalind E. M. Rickaby and Emily Flashman*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00032","DOIUrl":"10.1021/acsbiomedchemau.2c00032","url":null,"abstract":"<p >All aerobic organisms require O₂ for survival. When their O₂ is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O₂-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O₂ availability and can therefore act as plant O₂ sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, <i>Marchantia polymorpha</i> (MpPCO), and the freshwater algae, <i>Klebsormidium nitens</i> (KnPCO), have a similar function as PCO enzymes from <i>Arabidopsis thaliana</i>. We report that MpPCO and KnPCO show O₂-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the <i>Arabidopsis</i> ERF-VIIs transcription factors. This work confirms functional and O₂-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O₂-sensing pathways in these organisms and suggesting PCO O₂-sensing function could be important throughout the plant kingdom.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 5","pages":"521–528"},"PeriodicalIF":0.0,"publicationDate":"2022-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585510/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40586705","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":"Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry","authors":"Angelo E. Andres,&nbsp;Andres Mariano,&nbsp;Digamber Rane and Blake R. Peterson*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00031","DOIUrl":"10.1021/acsbiomedchemau.2c00031","url":null,"abstract":"<p >Drugs such as paclitaxel (Taxol) that bind microtubules are widely used for the treatment of cancer. Measurements of the affinity and selectivity of these compounds for their targets are largely based on studies of purified proteins, and only a few quantitative methods for the analysis of interactions of small molecules with microtubules in living cells have been reported. We describe here a novel method for rapidly quantifying the affinities of compounds that bind polymerized tubulin in living HeLa cells. This method uses the fluorescent molecular probe Pacific Blue-GABA-Taxol in conjunction with verapamil to block cellular efflux. Under physiologically relevant conditions of 37 °C, this combination allowed quantification of equilibrium saturation binding of this probe to cellular microtubules (<i>K</i>_d = 1.7 μM) using flow cytometry. Competitive binding of the microtubule stabilizers paclitaxel (cellular <i>K</i>_i = 22 nM), docetaxel (cellular <i>K</i>_i = 16 nM), cabazitaxel (cellular <i>K</i>_i = 6 nM), and ixabepilone (cellular <i>K</i>_i = 10 nM) revealed intracellular affinities for microtubules that closely matched previously reported biochemical affinities. By including a cooperativity factor (α) for curve fitting of allosteric modulators, this probe also allowed quantification of binding (<i>K</i>_b) of the microtubule destabilizers colchicine (<i>K</i>_b = 80 nM, α = 0.08), vinblastine (<i>K</i>_b = 7 nM, α = 0.18), and maytansine (<i>K</i>_b = 3 nM, α = 0.21). Screening of this assay against 1008 NCI diversity compounds identified NSC 93427 as a novel microtubule destabilizer (<i>K</i>_b = 485 nM, α = 0.02), illustrating the potential of this approach for drug discovery.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 5","pages":"529–537"},"PeriodicalIF":0.0,"publicationDate":"2022-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/38/80/bg2c00031.PMC9585582.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40586704","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":"Characterization of LipS1 and LipS2 from Thermococcus kodakarensis: Proteins Annotated as Biotin Synthases, which Together Catalyze Formation of the Lipoyl Cofactor","authors":"Syam Sundar Neti,&nbsp;Debangsu Sil,&nbsp;Douglas M. Warui,&nbsp;Olga A. Esakova,&nbsp;Amy E. Solinski,&nbsp;Dante A. Serrano,&nbsp;Carsten Krebs* and Squire J. Booker*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00018","DOIUrl":"https://doi.org/10.1021/acsbiomedchemau.2c00018","url":null,"abstract":"<p >Lipoic acid is an eight-carbon sulfur-containing biomolecule that functions primarily as a cofactor in several multienzyme complexes. It is biosynthesized as an attachment to a specific lysyl residue on one of the subunits of these multienzyme complexes. In <i>Escherichia coli</i> and many other organisms, this biosynthetic pathway involves two dedicated proteins: octanoyltransferase (LipB) and lipoyl synthase (LipA). LipB transfers an <i>n</i>-octanoyl chain from the octanoyl-acyl carrier protein to the target lysyl residue, and then, LipA attaches two sulfur atoms (one at C6 and one at C8) to give the final lipoyl cofactor. All classical lipoyl synthases (LSs) are radical <i>S</i>-adenosylmethionine (SAM) enzymes, which use an [Fe₄S₄] cluster to reductively cleave SAM to generate a 5′-deoxyadenosyl 5′-radical. Classical LSs also contain a second [Fe₄S₄] cluster that serves as the source of both appended sulfur atoms. Recently, a novel pathway for generating the lipoyl cofactor was reported. This pathway replaces the canonical LS with two proteins, LipS1 and LipS2, which act together to catalyze formation of the lipoyl cofactor. In this work, we further characterize LipS1 and LipS2 biochemically and spectroscopically. Although LipS1 and LipS2 were previously annotated as biotin synthases, we show that both proteins, unlike <i>E. coli</i> biotin synthase, contain two [Fe₄S₄] clusters. We identify the cluster ligands to both iron–sulfur clusters in both proteins and show that LipS2 acts only on an octanoyl-containing substrate, while LipS1 acts only on an 8-mercaptooctanoyl-containing substrate. Therefore, similarly to <i>E. coli</i> biotin synthase and in contrast to <i>E. coli</i> LipA, sulfur attachment takes place initially at the terminal carbon (C8) and then at the C6 methylene carbon.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 5","pages":"509–520"},"PeriodicalIF":0.0,"publicationDate":"2022-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.2c00018","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72199321","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":"Azithromycin Protects Retinal Glia Against Oxidative Stress-Induced Morphological Changes, Inflammation, and Cell Death","authors":"Binapani Mahaling,&nbsp;Narendra Pandala,&nbsp;Heuy-Ching Wang and Erin B. Lavik*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00013","DOIUrl":"10.1021/acsbiomedchemau.2c00013","url":null,"abstract":"<p >The reactivity of retinal glia in response to oxidative stress has a significant effect on retinal pathobiology. The reactive glia change their morphology and secret cytokines and neurotoxic factors in response to oxidative stress associated with retinal neurovascular degeneration. Therefore, pharmacological intervention to protect glial health against oxidative stress is crucial for maintaining homeostasis and the normal function of the retina. In this study, we explored the effect of azithromycin, a macrolide antibiotic with antioxidant, immunomodulatory, anti-inflammatory, and neuroprotective properties against oxidative stress-induced morphological changes, inflammation, and cell death in retinal microglia and Müller glia. Oxidative stress was induced by H₂O₂, and the intracellular oxidative stress was measured by DCFDA and DHE staining. The change in morphological characteristics such as the surface area, perimeter, and circularity was calculated using ImageJ software. Inflammation was measured by enzyme-linked immunosorbent assays for TNF-α, IL-1β, and IL-6. Reactive gliosis was characterized by anti-GFAP immunostaining. Cell death was measured by MTT assay, acridine orange/propidium iodide, and trypan blue staining. Pretreatment of azithromycin inhibits H₂O₂-induced oxidative stress in microglial (BV-2) and Müller glial (MIO-M1) cells. We observed that azithromycin inhibits oxidative stress-induced morphological changes, including the cell surface area, circularity, and perimeter in BV-2 and MIO-M1 cells. It also inhibits inflammation and cell death in both the glial cells. Azithromycin could be used as a pharmacological intervention on maintaining retinal glial health during oxidative stress.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 5","pages":"499–508"},"PeriodicalIF":0.0,"publicationDate":"2022-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/a8/92/bg2c00013.PMC10125304.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9349338","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":"Sphingosine Kinase 2 Inhibitors: Rigid Aliphatic Tail Derivatives Deliver Potent and Selective Analogues","authors":"Srinath Pashikanti,&nbsp;Daniel J. Foster,&nbsp;Yugesh Kharel,&nbsp;Anne M. Brown,&nbsp;David R. Bevan,&nbsp;Kevin R. Lynch and Webster L. Santos*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00017","DOIUrl":"10.1021/acsbiomedchemau.2c00017","url":null,"abstract":"<p >Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that interacts with five native G-protein coupled receptors (S1P1–5) to regulate cell growth, survival, and proliferation. S1P has been implicated in a variety of pathologies including cancer, kidney fibrosis, and multiple sclerosis. As key mediators in the synthesis of S1P, sphingosine kinase (SphK) isoforms 1 and 2 have attracted attention as viable targets for pharmacologic intervention. In this report, we describe the design, synthesis, and biological evaluation of sphingosine kinase 2 (SphK2) inhibitors with a focus on systematically introducing rigid structures in the aliphatic lipid tail present in existing SphK2 inhibitors. Experimental as well as molecular modeling studies suggest that conformationally restricted “lipophilic tail” analogues bearing a bulky terminal moiety or an internal phenyl ring are useful to complement the “J”-shaped sphingosine binding pocket of SphK2. We identified <b>14c</b> (SLP9101555) as a potent SphK2 inhibitor (<i>K</i>_i = 90 nM) with 200-fold selectivity over SphK1. Molecular docking studies indicated key interactions: the cyclohexyl ring binding in the cleft deep in the pocket, a trifluoromethyl group fitting in a small side cavity, and a hydrogen bond between the guanidino group and Asp308 (amino acid numbering refers to human SphK2 (isoform c) orthologue). <i>In vitro</i> studies using U937 human histiocytic lymphoma cells showed marked decreases in extracellular S1P levels in response to our SphK2 inhibitors. Administration of <b>14c</b> (dose: 5 mg/kg) to mice resulted in a sustained increase of circulating S1P levels, suggesting target engagement.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 5","pages":"469–489"},"PeriodicalIF":0.0,"publicationDate":"2022-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/c7/42/bg2c00017.PMC9585524.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40586706","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":"Investigation of Acid–Base Catalysis in Halimadienyl Diphosphate Synthase Involved in Mycobacterium tuberculosis Virulence","authors":"Cody Lemke,&nbsp;Kristin Roach,&nbsp;Teresa Ortega,&nbsp;Dean J. Tantillo,&nbsp;Justin B. Siegel and Reuben J. Peters*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00023","DOIUrl":"https://doi.org/10.1021/acsbiomedchemau.2c00023","url":null,"abstract":"<p >The devastating human pathogen<i>Mycobacterium tuberculosis</i> (Mtb) is able to parasitize phagosomal compartments within alveolar macrophage cells due, in part, to the activity of its cell-surface lipids. Prominent among these is 1-tuberculosinyl-adenosine (1-TbAd), a derivative of the diterpenoid tuberculosinyl (halima-5,13-dienyl) diphosphate produced by the class II diterpene cyclase encoded by Rv3377c, termed here MtHPS. Given the demonstrated ability of 1-TbAd to act as a virulence factor for Mtb and the necessity for Rv3377c for its production, there is significant interest in MtHPS activity. Class II diterpene cyclases catalyze a general acid–base-mediated carbocation cascade reaction initiated by protonation of the terminal alkene in the general diterpenoid precursor (<i>E,E,E</i>)-geranylgeranyl diphosphate and terminated by deprotonation of the final cyclized (and sometimes also rearranged) intermediate. Here, structure-guided mutagenesis was applied to characterize the various residues contributing to activation of the enzymatic acid, as well as identify the enzymatic base in MtHPS. Particularly given the ability of conservative substitution for the enzymatic base (Y479F) to generate an alternative product (labda-7,13-dienyl diphosphate) via deprotonation of an earlier unrearranged intermediate, further mutational analysis was carried out to introduce potential alternative catalytic bases. The results were combined with mechanistic molecular modeling to elucidate how these mutations affect the catalytic activity of this important enzyme. This not only provided detailed structure–function insight into MtHPS but also further emphasized the inert nature of the active site of MtHPS and class II diterpene cyclases more generally.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 5","pages":"490–498"},"PeriodicalIF":0.0,"publicationDate":"2022-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.2c00023","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72199475","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":"No Limits: Using Art to Inspire and Encourage Minorities to Pursue Careers in STEAM (STEM + Art)","authors":"Jayde Frederick*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00028","DOIUrl":"10.1021/acsbiomedchemau.2c00028","url":null,"abstract":"","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 3","pages":"171–172"},"PeriodicalIF":0.0,"publicationDate":"2022-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/01/e8/bg2c00028.PMC10114618.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9707395","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 Vitro Demonstration of Human Lipoyl Synthase Catalytic Activity in the Presence of NFU1","authors":"Douglas M. Warui,&nbsp;Debangsu Sil,&nbsp;Kyung-Hoon Lee,&nbsp;Syam Sundar Neti,&nbsp;Olga A. Esakova,&nbsp;Hayley L. Knox,&nbsp;Carsten Krebs* and Squire J. Booker*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00020","DOIUrl":"10.1021/acsbiomedchemau.2c00020","url":null,"abstract":"<p >Lipoyl synthase (LS) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of sulfur atoms at C6 and C8 of an <i>n</i>-octanoyllysyl side chain of a lipoyl carrier protein (LCP). The protein is a member of the radical <i>S</i>-adenosylmethionine (SAM) superfamily of enzymes, which use SAM as a precursor to a 5′-deoxyadenosyl 5′-radical (5′-dA·). The role of the 5′-dA· in the LS reaction is to abstract hydrogen atoms from C6 and C8 of the octanoyl moiety of the substrate to initiate subsequent sulfur attachment. All radical SAM enzymes have at least one [4Fe–4S] cluster that is used in the reductive cleavage of SAM to generate the 5′-dA·; however, LSs contain an additional auxiliary [4Fe–4S] cluster from which sulfur atoms are extracted during turnover, leading to degradation of the cluster. Therefore, these enzymes catalyze only 1 turnover in the absence of a system that restores the auxiliary cluster. In <i>Escherichia coli</i>, the auxiliary cluster of LS can be regenerated by the iron–sulfur (Fe–S) cluster carrier protein NfuA as fast as catalysis takes place, and less efficiently by IscU. NFU1 is the human ortholog of <i>E. coli</i> NfuA and has been shown to interact directly with human LS (i.e., LIAS) in yeast two-hybrid analyses. Herein, we show that NFU1 and LIAS form a tight complex in vitro and that NFU1 can efficiently restore the auxiliary cluster of LIAS during turnover. We also show that BOLA3, previously identified as being critical in the biosynthesis of the lipoyl cofactor in humans and <i>Saccharomyces cerevisiae</i>, has no direct effect on Fe–S cluster transfer from NFU1 or GLRX5 to LIAS. Further, we show that ISCA1 and ISCA2 can enhance LIAS turnover, but only slightly.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 5","pages":"456–468"},"PeriodicalIF":0.0,"publicationDate":"2022-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585516/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40586707","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}