{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T01:30:49Z","timestamp":1790904649085,"version":"4.1.0"},"reference-count":65,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,4,15]],"date-time":"2024-04-15T00:00:00Z","timestamp":1713139200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Tuberculosis (TB) stands as the second most fatal infectious disease globally, causing 1.3 million deaths in 2022. The resurgence of TB and the alarming rise of antibiotic resistance demand urgent call to develop novel antituberculosis drugs. Despite concerted efforts to control TB, the disease persists and spreads rapidly on a global scale. Targeting stress response pathways in <jats:italic>Mycobacterium tuberculosis (Mtb)<\/jats:italic> has become imperative to achieve complete eradication. This study employs subtractive genomics to identify and prioritize potential drug targets among the hypothetical proteins of <jats:italic>Mtb<\/jats:italic>, focusing on indispensable pathways. Amongst 177 essential hypothetical proteins, 152 were nonhomologous to human. These proteins participated in 34 pathways, and a 20-fold enrichment of SUF pathway genes led to its selection as a target pathway. Fe\u2013S clusters are fundamental, widely distributed protein cofactors involved in vital cellular processes. The survival of <jats:italic>Mtb<\/jats:italic> in a hypoxic environment relies on the iron\u2013sulfur (Fe\u2013S) cluster biogenesis pathway for the repair of damaged Fe\u2013S clusters. It also protects pathogen against drugs, ensuring controlled iron utilization and contributing to drug resistance. In <jats:italic>Mtb<\/jats:italic>, six proteins of Fe\u2013S cluster assembly pathway are encoded by the <jats:italic>suf<\/jats:italic> operon. The present study was focused on SufD because of its role in iron acquisition and prevention of Fenton reaction. The research further delves into the <jats:italic>in silico<\/jats:italic> characterization of SufD, utilizing bioinformatics tools for sequence and structure based analysis. The protein\u2019s structural features, including the identification of conserved regions, motifs, and 3D structure prediction enhanced functional annotation. Target based virtual screening of compounds from the ChEMBL database resulted in 12 inhibitors with best binding affinities. Drug likeness and ADMET profiling of potential inhibitors identified promising compounds with favorable drug-like properties. The study also involved cloning in SUMO-pRSF-Duet1 expression vector, overexpression, and purification of recombinant SufD from <jats:italic>E. coli<\/jats:italic> BL21 (DE3) cells. Optimization of expression conditions resulted in soluble production, and subsequent purification highlighting the efficacy of the SUMO fusion system for challenging <jats:italic>Mtb<\/jats:italic> proteins in <jats:italic>E. coli<\/jats:italic>. These findings provide valuable insights into pharmacological targets for future experimental studies, holding promise for the development of targeted therapy against <jats:italic>Mtb<\/jats:italic>.<\/jats:p>","DOI":"10.3389\/fmicb.2024.1369645","type":"journal-article","created":{"date-parts":[[2024,4,15]],"date-time":"2024-04-15T16:12:55Z","timestamp":1713197575000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":12,"title":["Genome mining of Mycobacterium tuberculosis: targeting SufD as a novel drug candidate through in silico characterization and inhibitor screening"],"prefix":"10.3389","volume":"15","author":[{"given":"Neelima","family":"Gorityala","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony Samit","family":"Baidya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Someswar R.","family":"Sagurthi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2024,4,15]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"298","DOI":"10.1002\/path.2055","article-title":"Oxygen status of lung granulomas in Mycobacterium tuberculosis-infected mice","volume":"210","author":"Aly","year":"2006","journal-title":"J. Pathol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"160","DOI":"10.1016\/j.jiph.2020.12.005","article-title":"Identification of potential drug targets in human pathogen Bacillus cereus and insight for finding inhibitor through subtractive proteome and molecular docking studies","volume":"14","author":"Anis Ahamed","year":"2021","journal-title":"J. Infect. Public Health"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1128\/MMBR.00034-07","article-title":"Fe-S cluster assembly pathways in bacteria","volume":"72","author":"Ayala-Castro","year":"2008","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"W39","DOI":"10.1093\/nar\/gkv416","article-title":"The MEME Suite","volume":"43","author":"Bailey","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"e1003902","DOI":"10.1371\/journal.ppat.1003902","article-title":"Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection","volume":"10","author":"Bhaskar","year":"2014","journal-title":"PLoS Pathog."},{"key":"ref6","volume-title":"Discovery studio, release 2020","author":"Biovia","year":"2020"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"435","DOI":"10.1002\/cmdc.200700139","article-title":"Lessons learnt from assembling screening libraries for drug discovery for neglected diseases","volume":"3","author":"Brenk","year":"2008","journal-title":"ChemMedChem"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"W402","DOI":"10.1093\/nar\/gkz297","article-title":"The PSIPRED protein analysis workbench: 20 years on","volume":"47","author":"Buchan","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.pep.2005.03.016","article-title":"SUMO fusion technology for difficult-to-express proteins","volume":"43","author":"Butt","year":"2005","journal-title":"Protein Expr. Purif."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"1351","DOI":"10.1016\/j.chembiol.2016.09.012","article-title":"A small-molecule inhibitor of Iron-sulfur cluster assembly uncovers a link between virulence regulation and metabolism in Staphylococcus aureus","volume":"23","author":"Choby","year":"2016","journal-title":"Cell Chem. Biol."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"952","DOI":"10.1128\/jb.184.4.952-961.2002","article-title":"Identification of virulence genes in a pathogenic strain of Pseudomonas aeruginosa by representational difference analysis","volume":"184","author":"Choi","year":"2002","journal-title":"J. Bacteriol."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"10881","DOI":"10.1093\/nar\/16.22.10881","article-title":"Multiple sequence alignment with hierarchical clustering","volume":"16","author":"Corpet","year":"1988","journal-title":"Nucleic Acids Res."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"e02133","DOI":"10.1128\/mBio.02133-16","article-title":"Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis","volume":"8","author":"DeJesus","year":"2017","journal-title":"MBio"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"732","DOI":"10.3390\/biom13050732","article-title":"Structural and biochemical characterization of Mycobacterium tuberculosis zinc SufU-SufS complex","volume":"13","author":"Elchennawi","year":"2023","journal-title":"Biomol. Ther."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"174","DOI":"10.3390\/inorganics10100174","article-title":"Iron\u2013sulfur clusters toward stresses: implication for understanding and fighting tuberculosis","volume":"10","author":"Elchennawi","year":"2022","journal-title":"Inorganics"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"1157","DOI":"10.1016\/j.drudis.2019.03.015","article-title":"ADMET modeling approaches in drug discovery","volume":"24","author":"Ferreira","year":"2019","journal-title":"Drug Discov. Today"},{"key":"ref17","doi-asserted-by":"publisher","first-page":"171","DOI":"10.1038\/nchembio0406-171","article-title":"Iron-sulfur clusters: ever-expanding roles","volume":"2","author":"Fontecave","year":"2006","journal-title":"Nat. Chem. Biol. Apr."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"1564","DOI":"10.1038\/s41559-022-01857-1","article-title":"An early origin of iron\u2013sulfur cluster biosynthesis machineries before earth oxygenation","volume":"6","author":"Garcia","year":"2022","journal-title":"Nat. Ecol. Evol."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"2628","DOI":"10.1093\/bioinformatics\/btz931","article-title":"ShinyGO: a graphical gene-set enrichment tool for animals and plants","volume":"36","author":"Ge","year":"2020","journal-title":"Bioinformatics"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"230","DOI":"10.22159\/ajpcr.2018.v11i3.22105","article-title":"In silico identification of novel drug targets in acinetobacter baumannii by subtractive genomic approach","volume":"11","author":"Goyal","year":"2018","journal-title":"Asian J. Pharm. Clin. Res."},{"key":"ref9001","doi-asserted-by":"publisher","first-page":"e61","DOI":"10.1371\/journal.pcbi.0020061","article-title":"Prioritizing Genomic Drug Targets in Pathogens: Application to Mycobacterium tuberculosis","volume":"2","author":"Hasan","year":"2006","journal-title":"PLOS Computational Biology"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"29717","DOI":"10.1074\/jbc.M115.680934","article-title":"Functional dynamics revealed by the structure of the SufBCD complex, a novel ATP-binding cassette (ABC) protein that serves as a scaffold for Iron-sulfur cluster biogenesis","volume":"290","author":"Hirabayashi","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"6137","DOI":"10.1128\/JB.187.17.6137-6146.2005","article-title":"Identification of the Mycobacterium tuberculosis SUF machinery as the exclusive mycobacterial system of [Fe-S] cluster assembly: evidence for its implication in the Pathogen\u2019s survival","volume":"187","author":"Huet","year":"2005","journal-title":"J. Bacteriol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1073","DOI":"10.1111\/j.1365-2958.2006.05028.x","article-title":"Iron-Sulphur clusters and the problem with oxygen","volume":"59","author":"Imlay","year":"2006","journal-title":"Mol. Microbiol."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"929","DOI":"10.1074\/jbc","article-title":"Micromolar intracellular hydrogen peroxide disrupts metabolism by damaging iron-sulfur enzymes","volume":"282","author":"Jang","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1186\/1471-2105-15-S16-S7","article-title":"Bhageerath-H: a homology\/ab initio hybrid server for predicting tertiary structures of monomeric soluble proteins","volume":"15","author":"Jayaram","year":"2014","journal-title":"BMC Bioinform."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"6195","DOI":"10.1093\/nar\/gkl789","article-title":"Bhageerath: an energy based web enabled computer software suite for limiting the search space of tertiary structures of small globular proteins","volume":"34","author":"Jayaram","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"176","DOI":"10.1007\/s12275-020-9525-5","article-title":"Microbial community analysis using high-throughput sequencing technology: a beginner\u2019s guide for microbiologists","volume":"58","author":"Jo","year":"2020","journal-title":"J. Microbiol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"222","DOI":"10.1186\/1471-2164-8-222","article-title":"Quantitative assessment of relationship between sequence similarity and function similarity","volume":"8","author":"Joshi","year":"2007","journal-title":"BMC Genomics"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"457","DOI":"10.1016\/j.chom.2012.03.008","article-title":"Host cell autophagy activated by antibiotics is required for their effective Antimycobacterial drug action","volume":"11","author":"Kim","year":"2012","journal-title":"Cell Host Microbe"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"797","DOI":"10.1016\/j.cell.2007.06.049","article-title":"A common mechanism of cellular death induced by bactericidal antibiotics","volume":"130","author":"Kohanski","year":"2007","journal-title":"Cell"},{"key":"ref31","doi-asserted-by":"publisher","first-page":"e0349622","DOI":"10.1128\/mbio.03496-22","article-title":"Adjunctive integrated stress response inhibition accelerates tuberculosis clearance in mice","volume":"14","author":"Krug","year":"2023","journal-title":"MBio"},{"key":"ref32","doi-asserted-by":"publisher","first-page":"283","DOI":"10.1107\/S0021889892009944","article-title":"PROCHECK: a program to check the stereochemical quality of protein structures","volume":"26","author":"Laskowski","year":"1993","journal-title":"J. Appl. Crystallogr."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"W89","DOI":"10.1093\/nar\/gki414","article-title":"Pro Func: a server for predicting protein function from 3D structure","volume":"33","author":"Laskowski","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"D677","DOI":"10.1093\/nar\/gkaa917","article-title":"DEG 15, an update of the database of essential genes that includes built-in analysis tools","volume":"49","author":"Luo","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref35","year":"2011"},{"key":"ref36","doi-asserted-by":"publisher","DOI":"10.1093\/femspd\/fty036","article-title":"Host-pathogen redox dynamics modulate Mycobacterium tuberculosis pathogenesis","volume":"76","author":"Pacl","year":"2018","journal-title":"Pathog. Dis."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"17359","DOI":"10.1038\/s41598-018-35012-3","article-title":"Iron homeostasis in Mycobacterium tuberculosis is essential for persistence","volume":"8","author":"Pandey","year":"2018","journal-title":"Sci. Rep."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"436","DOI":"10.1038\/nrmicro2356","article-title":"Building Fe\u2013S proteins: bacterial strategies","volume":"8","author":"Py","year":"2010","journal-title":"Nat. Rev. Microbiol."},{"key":"ref9002","doi-asserted-by":"publisher","first-page":"e00100","DOI":"10.1128\/IAI.00100-17","article-title":"The Suf Iron-Sulfur Cluster Biosynthetic System Is Essential in Staphylococcus aureus, and Decreased Suf Function Results in Global Metabolic Defects and Reduced Survival in Human Neutrophils","volume":"85","author":"Roberts","year":"2017","journal-title":"Infect. Immun."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"725","DOI":"10.1038\/nprot.2010.5","article-title":"I-TASSER: a unified platform for automated protein structure and function prediction","volume":"5","author":"Roy","year":"2010","journal-title":"Nat. Protoc."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"9402","DOI":"10.1021\/bi1011546","article-title":"SufD and SufC ATPase activity are required for Iron acquisition during in vivo Fe-S cluster formation on SufB","volume":"49","author":"Saini","year":"2010","journal-title":"Biochemistry"},{"key":"ref41","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1046\/j.1365-2958.2003.03425.x","article-title":"Genes required for mycobacterial growth defined by high density mutagenesis","volume":"48","author":"Sassetti","year":"2003","journal-title":"Mol. Microbiol."},{"key":"ref42","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1084\/jem.20030846","article-title":"Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment","volume":"198","author":"Schnappinger","year":"2003","journal-title":"J. Exp. Med."},{"key":"ref43","volume-title":"Schr\u00f6dinger Release 2023\u20134: LigPrep","year":"2024"},{"key":"ref44","doi-asserted-by":"publisher","first-page":"649","DOI":"10.1042\/EBC20190042","article-title":"Uncovering protein structure","volume":"64","author":"Stollar","year":"2020","journal-title":"Essays Biochem."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"W314","DOI":"10.1093\/nar\/gkt503","article-title":"Depth: a web server to compute depth, cavity sizes, detect potential small-molecule ligand-binding cavities and predict the pKa of ionizable residues in proteins","volume":"41","author":"Tan","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"3315","DOI":"10.1021\/jm051197e","article-title":"MolDock: a new technique for high-accuracy molecular docking","volume":"49","author":"Thomsen","year":"2006","journal-title":"J. Med. Chem."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"W363","DOI":"10.1093\/nar\/gky473","article-title":"CASTp 3.0: computed atlas of surface topography of proteins","volume":"46","author":"Tian","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"e1010475","DOI":"10.1371\/journal.ppat.1010475","article-title":"Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation, metabolism, and survival in vivo","volume":"18","author":"Tripathi","year":"2022","journal-title":"PLoS Pathog."},{"key":"ref49","year":""},{"key":"ref50","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1016\/j.compbiolchem.2013.11.005","article-title":"Identification and characterization of potential drug targets by subtractive genome analyses of methicillin resistant Staphylococcus aureus","volume":"48","author":"Uddin","year":"2014","journal-title":"Comput. Biol. Chem."},{"key":"ref51","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1155\/2017\/3647657","article-title":"Fe-S clusters emerging as targets of therapeutic drugs","volume":"2017","author":"Vernis","year":"2017","journal-title":"Oxidative Med. Cell. Longev."},{"key":"ref52","doi-asserted-by":"crossref","first-page":"2333","DOI":"10.1128\/IAI.01515-07","article-title":"Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates","volume":"76","author":"Via","year":"2008","journal-title":"Infect. Immun."},{"key":"ref53","doi-asserted-by":"publisher","first-page":"1881","DOI":"10.1038\/ncomms2898","article-title":"Mycobacterium tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction","volume":"4","author":"Vilch\u00e8ze","year":"2013","journal-title":"Nat. Commun."},{"key":"ref54","doi-asserted-by":"publisher","first-page":"105","DOI":"10.3389\/fmicb.2011.00105","article-title":"The response of Mycobacterium tuberculosis to reactive oxygen and nitrogen species","volume":"2","author":"Voskuil","year":"2011","journal-title":"Front. Microbiol."},{"key":"ref55","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1016\/j.micpath.2017.12.063","article-title":"Subtractive genome analysis for in silico identification and characterization of novel drug targets in Streptococcus pneumonia strain JJA","volume":"115","author":"Wadood","year":"2018","journal-title":"Microb. Pathog."},{"key":"ref56","doi-asserted-by":"publisher","first-page":"e0200145","DOI":"10.1371\/journal.pone.0200145","article-title":"Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis","volume":"13","author":"Willemse","year":"2018","journal-title":"PLoS One"},{"key":"ref57","doi-asserted-by":"publisher","first-page":"1067","DOI":"10.1093\/bioinformatics\/bty707","article-title":"Yun tang, admetSAR 2.0: web-service for prediction and optimization of chemical ADMET properties","volume":"35","author":"Yang","year":"2019","journal-title":"Bioinformatics"},{"key":"ref58","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1038\/nmeth.3213","article-title":"The I-TASSER Suite: protein structure and function prediction","volume":"12","author":"Yang","year":"2015","journal-title":"Nat. Methods"},{"key":"ref59","doi-asserted-by":"publisher","first-page":"394","DOI":"10.1186\/s12864-019-5746-6","article-title":"Investigating function roles of hypothetical proteins encoded by the Mycobacterium tuberculosis H37Rv genome","volume":"20","author":"Yang","year":"2019","journal-title":"BMC Genomics"},{"key":"ref60","doi-asserted-by":"publisher","first-page":"99368","DOI":"10.1371\/journal.pone.0099368","article-title":"CELLO2GO: a web server for protein subCELlularLOcalization prediction with functional gene ontology annotation","volume":"9","author":"Yu","year":"2014","journal-title":"PLoS One"},{"key":"ref61","doi-asserted-by":"publisher","first-page":"1608","DOI":"10.1093\/bioinformatics\/btq249","article-title":"PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes","volume":"26","author":"Yu","year":"2010","journal-title":"Bioinformatics"},{"key":"ref62","doi-asserted-by":"publisher","first-page":"9387","DOI":"10.1038\/s41598-017-09846-2","article-title":"Mapping the key residues of SufB and SufD essential for biosynthesis of iron-sulfur clusters","volume":"7","author":"Yuda","year":"2017","journal-title":"Sci. Rep."},{"key":"ref63","doi-asserted-by":"publisher","first-page":"271D","DOI":"10.1093\/nar\/gkh024","article-title":"DEG: a database of essential genes","volume":"32","author":"Zhang","year":"2004","journal-title":"Nucleic Acids Res."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2024.1369645\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,4,15]],"date-time":"2024-04-15T16:13:02Z","timestamp":1713197582000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2024.1369645\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,4,15]]},"references-count":65,"alternative-id":["10.3389\/fmicb.2024.1369645"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2024.1369645","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,4,15]]},"article-number":"1369645"}}