{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T04:02:44Z","timestamp":1790481764810,"version":"4.1.0"},"reference-count":72,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2024,11,27]],"date-time":"2024-11-27T00:00:00Z","timestamp":1732665600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2024,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Human neutrophil elastase (hNE), a serine protease released by neutrophils during inflammation, plays a major role in the pathophysiology of several conditions especially in inflammatory lung diseases. Its inhibition constitutes, therefore, a promising therapeutic strategy to combat these diseases. In this work, we characterized the in vitro properties of a VHH (i.e., the antigen binding domain of camelid heavy chain\u2010only antibodies), referred to as NbE201. This VHH is able to inhibit tightly, selectively and competitively both human and murine elastases with the inhibition constants (<jats:italic>K<\/jats:italic><jats:sub><jats:italic>i<\/jats:italic><\/jats:sub>) of 4.1\u2009\u00b1\u20090.9\u2009nM and 36.8\u2009\u00b1\u20093.9\u2009nM, respectively. The IC<jats:sub>50<\/jats:sub> for the inhibition of the hydrolysis of elastin is in the same range to that of alpha\u20101 antitrypsin (i.e., the main endogenous inhibitor of hNE also used in the clinic) and 14 times better than that of Sivelestat (i.e., the 2nd clinically approved hNE inhibitor). The X\u2010ray crystal structure of the NbE201\u2010hNE complex reveals that the Complementarity Determining Regions CDR1 and CDR3 of the VHH bind into the substrate binding pocket of hNE and prevent the access to small or macromolecular substrates. They do not, however, bind deep enough into the pocket to be hydrolyzed. NbE201 is highly stable towards oxidation, deamidation, and chemical or thermal denaturation. NbE201 is therefore likely to tolerate manufacturing processes during drug development. These results highlight the high potential of NbE201 as a (pre)clinical tool to diagnose and treat diseases associated with excessive hNE activity, and for fundamental research to better understand the role of hNE in these conditions.<\/jats:p>","DOI":"10.1002\/pro.5227","type":"journal-article","created":{"date-parts":[[2024,11,28]],"date-time":"2024-11-28T05:34:22Z","timestamp":1732772062000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Enzymatic, structural, and biophysical characterization of a single\u2010domain antibody (VHH) selectively and tightly inhibiting neutrophil elastase and exhibiting favorable developability properties"],"prefix":"10.1002","volume":"33","author":[{"given":"Paola","family":"Redeghieri","sequence":"first","affiliation":[{"name":"Nano\u2010Antibodies to Explore Protein Structure and Functions (NEPTUNS) Centre for Protein Engineering, InBios, Department of Life Sciences, University of Li\u00e8ge  Li\u00e8ge Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jo\u00ebl","family":"Moray","sequence":"additional","affiliation":[{"name":"Nano\u2010Antibodies to Explore Protein Structure and Functions (NEPTUNS) Centre for Protein Engineering, InBios, Department of Life Sciences, University of Li\u00e8ge  Li\u00e8ge Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fr\u00e9d\u00e9ric","family":"Kerff","sequence":"additional","affiliation":[{"name":"Biological Macromolecule Crystallography, Centre for Protein Engineering, InBios, Department of Life Sciences University of Li\u00e8ge  Li\u00e8ge Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sophie","family":"Gohy","sequence":"additional","affiliation":[{"name":"Department of Pneumology, ENT and Dermatology, Institute of Experimental and Clinical Research (IREC) Universit\u00e9 Catholique de Louvain  Brussels Belgium"},{"name":"Cystic Fibrosis Reference Centre Cliniques Universitaires Saint\u2010Luc  Brussels Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Teresinha","family":"Leal","sequence":"additional","affiliation":[{"name":"Louvain Center for Toxicology and Applied Pharmacology (LTAP), Institute of Experimental and Clinical Research (IREC) Universit\u00e9 Catholique de Louvain  Brussels Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Serge","family":"Muyldermans","sequence":"additional","affiliation":[{"name":"Laboratory of Cellular and Molecular Immunology Vrije Universiteit Brussel  Brussels Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0784-8771","authenticated-orcid":false,"given":"Rita","family":"Vanbever","sequence":"additional","affiliation":[{"name":"Louvain Drug Research Institute (LDRI) Universit\u00e9 Catholique de Louvain  Brussels Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco Javier","family":"Morales\u2010Y\u00e1nez","sequence":"additional","affiliation":[{"name":"Nano\u2010Antibodies to Explore Protein Structure and Functions (NEPTUNS) Centre for Protein Engineering, InBios, Department of Life Sciences, University of Li\u00e8ge  Li\u00e8ge Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mireille","family":"Dumoulin","sequence":"additional","affiliation":[{"name":"Nano\u2010Antibodies to Explore Protein Structure and Functions (NEPTUNS) Centre for Protein Engineering, InBios, Department of Life Sciences, University of Li\u00e8ge  Li\u00e8ge Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,11,27]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.632687"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/0014-5793(88)80118-2"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0253364"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41385-019-0235-4"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1165\/ajrcmb\/9.4.455"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444904016427"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1986.tb04521.x"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI109364"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/prp2.535"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1128\/aac.01499-22"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1183\/13993003.01050-2024"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biopha.2020.111152"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms222011136"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.45.10.2807-2812.2001"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471220639"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.2147\/JIR.S234710"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jim.2008.04.010"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-23167-z"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.3390\/ph14100979"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1038\/nsb0996-803"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jaci.2017.04.022"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1007\/s44231-022-00012-5"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcmgh.2021.03.002"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1110\/ps.34602"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.1152\/ajpgi.00393.2015"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2008.11.046"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444910007493"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2008.05.009"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-016-7889-6"},{"key":"e_1_2_8_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0014-5793(96)01508-6"},{"key":"e_1_2_8_32_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2023.102969"},{"key":"e_1_2_8_33_1","doi-asserted-by":"publisher","DOI":"10.1161\/ATVBAHA.116.308726"},{"key":"e_1_2_8_34_1","first-page":"49","article-title":"Lysosomal enzymes from polymorphonuclear leukocytes and proteinase inhibitors in patients with cystic fibrosis","volume":"134","author":"Goldstein W","year":"1986","journal-title":"Am Rev Respir Dis"},{"key":"e_1_2_8_35_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12931-017-0691-x"},{"key":"e_1_2_8_36_1","doi-asserted-by":"publisher","DOI":"10.1111\/1523-1747.ep12342990"},{"key":"e_1_2_8_37_1","doi-asserted-by":"publisher","DOI":"10.1111\/all.14746"},{"key":"e_1_2_8_38_1","doi-asserted-by":"publisher","DOI":"10.18632\/oncotarget.1631"},{"key":"e_1_2_8_39_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1142"},{"key":"e_1_2_8_40_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00432-022-04310-9"},{"key":"e_1_2_8_41_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-08-027379-2.50034-X"},{"key":"e_1_2_8_42_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejphar.2020.173326"},{"key":"e_1_2_8_43_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.0c00493"},{"key":"e_1_2_8_44_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444909047337"},{"key":"e_1_2_8_45_1","doi-asserted-by":"publisher","DOI":"10.1016\/0006-291X(91)91862-7"},{"key":"e_1_2_8_46_1","doi-asserted-by":"publisher","DOI":"10.1080\/14737159.2017.1374174"},{"key":"e_1_2_8_47_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0909049507060670"},{"key":"e_1_2_8_48_1","doi-asserted-by":"publisher","DOI":"10.1124\/pr.110.002733"},{"key":"e_1_2_8_49_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M116.732503"},{"key":"e_1_2_8_50_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja5130786"},{"key":"e_1_2_8_51_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejps.2023.106522"},{"key":"e_1_2_8_52_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2023.104889"},{"key":"e_1_2_8_53_1","doi-asserted-by":"publisher","DOI":"10.1183\/16000617.0001-2024"},{"key":"e_1_2_8_54_1","doi-asserted-by":"publisher","DOI":"10.1080\/13543784.2023.2263366"},{"key":"e_1_2_8_55_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0021889807021206"},{"key":"e_1_2_8_56_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-063011-092449"},{"key":"e_1_2_8_57_1","doi-asserted-by":"publisher","DOI":"10.1016\/0076-6879(86)31045-0"},{"key":"e_1_2_8_58_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biochi.2015.01.012"},{"key":"e_1_2_8_59_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chest.2017.03.056"},{"key":"e_1_2_8_60_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA119.011712"},{"key":"e_1_2_8_61_1","doi-asserted-by":"publisher","DOI":"10.1002\/adfm.202101633"},{"key":"e_1_2_8_62_1","doi-asserted-by":"publisher","DOI":"10.1016\/0006-291X(68)90326-4"},{"key":"e_1_2_8_63_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2011.11.036"},{"key":"e_1_2_8_64_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R100016200"},{"key":"e_1_2_8_65_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.3285470"},{"key":"e_1_2_8_66_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)61505-X"},{"key":"e_1_2_8_67_1","doi-asserted-by":"publisher","DOI":"10.1155\/2015\/293053"},{"key":"e_1_2_8_68_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)67784-7"},{"key":"e_1_2_8_69_1","unstructured":"U.S. Food and Drug Administration.FDA Sivelestat.https:\/\/www.accessdata.fda.gov\/scripts\/opdlisting\/oopd\/detailedIndex.cfm?cfgridkey=7960202021."},{"key":"e_1_2_8_70_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmcl.2015.08.049"},{"key":"e_1_2_8_71_1","doi-asserted-by":"publisher","DOI":"10.1021\/bc300066a"},{"key":"e_1_2_8_72_1","doi-asserted-by":"publisher","DOI":"10.1097\/01.CCM.0000133332.48386.85"},{"key":"e_1_2_8_73_1","doi-asserted-by":"publisher","DOI":"10.1002\/ski2.90"}],"container-title":["Protein Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pro.5227","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,12,9]],"date-time":"2024-12-09T14:01:56Z","timestamp":1733752916000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/pro.5227"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11,27]]},"references-count":72,"journal-issue":{"issue":"12","published-print":{"date-parts":[[2024,12]]}},"alternative-id":["10.1002\/pro.5227"],"URL":"https:\/\/doi.org\/10.1002\/pro.5227","archive":["Portico"],"relation":{},"ISSN":["0961-8368","1469-896X"],"issn-type":[{"value":"0961-8368","type":"print"},{"value":"1469-896X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,11,27]]},"assertion":[{"value":"2024-05-10","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2024-10-28","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2024-11-27","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e5227"}}