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Peptides were first loaded on the trap column (Acclaim PepMap 100 C18, 75 µm ID x 2 cm, 3 µm particle size, Thermo Scientific), washed with 0.1% formic acid in water for 7 min at 5 µL/min and subsequently transferred to the separation column (IonOpticks Aurora C18 column, 25 cm × 75 µm, 1.7 µm) and separated over a 60 min gradient from 5% to 28% B, then to 40 % B over 13 min, followed by 10 min at 95% before re-equilibration and at a flow rate of 400 nL/min. The mobile phases A and B were 0.1 % (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile, respectively. The timsTOF Pro was operated in data-independent dia-PASEF mode with the dual TIMS analyser operating at equal accumulation and ramp times of 100 ms each with a set 1/K0 ion mobility range from 0.60 to 1.60 V × s/cm2 for MS1 scans. The dia-PASEF settings for fragmentation were set to a mass range of 400 to 1201 m/z and an ion mobility range of 0.60 to 1.43 V × s × cm-2. Two ion mobility isolation windows were performed per dia-PASEF scan with 26 m/z window widths. A total of 32 isolation windows with 1 m/z overlaps to cover the mass range were used resulting in 16 dia-PASEF scans per MS1 scan and an estimated total cycle time of 1.80 s (see Table). The collision energy was ramped linearly as a function of the mobility from 59 eV at 1/K0 = 1.3 V × s × cm-2 to 20 eV at 1/K0 = 0.85 V × s × cm-2. TIMS elution voltages were calibrated linearly to obtain the reduced ion mobility coefficients (1/K0) using three Agilent ESI-L Tuning Mix ions (m/z 622, 922 and 1,222) spiked on the CaptiveSpray Source inlet filter. The timsTOF Pro was operated in data-independent dia-PASEF mode with the dual TIMS analyser operating at equal accumulation and ramp times of 100 ms each with a set 1/K0 ion mobility range from 0.60 to 1.60 V × s/cm2 for MS1 scans. The dia-PASEF settings for fragmentation were set to a mass range of 400 to 1201 m/z and an ion mobility range of 0.60 to 1.43 V × s × cm-2. Two ion mobility isolation windows were performed per dia-PASEF scan with 26 m/z window widths. A total of 32 isolation windows with 1 m/z overlaps to cover the mass range were used resulting in 16 dia-PASEF scans per MS1 scan and an estimated total cycle time of 1.80 s (see Table). The collision energy was ramped linearly as a function of the mobility from 59 eV at 1/K0 = 1.3 V × s × cm-2 to 20 eV at 1/K0 = 0.85 V × s × cm-2. TIMS elution voltages were calibrated linearly to obtain the reduced ion mobility coefficients (1/K0) using three Agilent ESI-L Tuning Mix ions (m/z 622, 922 and 1,222) spiked on the CaptiveSpray Source inlet filter."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Data was acquired using Xcalibur software version 3.1 sp3 and proteomics raw data were analysed using MaxQuant12 (ver. 1.6.2.10) which uses the Andromeda search engine.13 Settings were default except for LFQ-quantification and match between runs, which were activated during search. The parameters can be found attached in the appendix. Perseus 2.0.11.0 was used for analysis.14 Data was filtered as following: Potential contaminants’ were removed as well as ‘reverse peptides’ and ‘only identified by site’. LFQ intensities were log2 transformed. Rows were annotated in groups – DMSO (control) and different probe treatments. Rows were filtered for 2 valid values in at least one group. Missing values were imputed from normal distribution. Annotations derived from data banks (Uniprot15) were added. Each condition was performed in biological quadruplicates (n = 5) and volcano plots were created based on two-sided two sample Student‘s t-tests."],"omics_type":["Proteomics"],"labhead":["Stephan Axel Sieber"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["TUM School of Natural Sciences, Department Biosciences, Chair of Organic Chemistry II, Center for Functional Protein Assemblies (CPA), Technical University Munich (TUM); Ernst-Otto-Fischer Str. 8, 85748 Garching, Germany"],"species":["Staphylococcus Schleiferi","Staphylococcus Pseudintermedius"],"publication":["41851492 Fiedler MK, Pandler MSI, Gong R, Fuchs S, Rox K, Friedrich V, Pfeiffer D, Singh D, Reinhardt T, Mibus C, Huber M, Hess CR, Mejías-Luque R, Gerhard M, Groll M, Sieber SA. Metronidazole and ether derivatives target Helicobacter pylori via simultaneous stress induction and inhibition. Nat Microbiol. 2026 11(4):1049-1063 10.1038/s41564-026-02291-w"],"submitter_mail":["marianne.pandler@tum.de"],"submitter_affiliation":["TUM"],"submitter_country":["Germany"],"pubmed_abstract":["Metronidazole is a front-line drug for the treatment of Helicobacter pylori infections. However, its mode of action and cellular targets are poorly defined, and higher dosing and combination therapies are required to overcome resistance. Here we performed activity-based protein profiling with tailored metronidazole probes and identified chaperonin HpGroEL and thiol peroxidase HpTpx as prominent targets, the latter being essential for H. pylori survival under oxidative stress. Alkynylated ether probes exhibited enhanced antibacterial potency compared with the parent drug in vitro, including activity against resistant strains. Biological assays, chemical proteomics and co-crystallization studies confirmed target engagement, with enhanced binding of ether derivatives to HpTpx. Refined ether analogues exhibited favourable pharmacological profiles without cytotoxicity. The in vivo activity of ether analogues using an H. pylori mouse model demonstrated full bacterial eradication at low dosing of 0.3 mg kg<sup>-1</sup> day<sup>-1</sup>. Our findings reveal that stress induction and simultaneous inhibition of the stress response represent a mechanism of this compound class."],"pubmed_title":["Metronidazole and ether derivatives target Helicobacter pylori via simultaneous stress induction and inhibition."],"pubmed_authors":["Fiedler Michaela K MK, Pandler Marianne S I MSI, Gong Ruolan R, Fuchs Sonja S, Rox Katharina K, Friedrich Verena V, Pfeiffer Dietmar D, Singh Dharmesh D, Reinhardt Till T, Mibus Cora C, Huber Matthias M, Hess Corinna R CR, Mejías-Luque Raquel R, Gerhard Markus M, Groll Michael M, Sieber Stephan A SA"],"additional_accession":[]},"is_claimable":false,"name":"Dual-targeting 5-nitroimidazole ethers eradicate H. pylori by induction of stress and disabling its adaptive response","description":"Metronidazole represents a front line-drug against Helicobacter pylori. To unravel its largely unknown cellular targets, we performed activity-based protein profiling (ABPP). Surprisingly, an alkynylated ether probe (Metro-P3) exhibited a 60-fold enhanced potency. Chaperonin HpGroEL and thiol peroxidase HpTpx were identified as prominent Metro-P3 targets, the latter of which is essential for H. pylori survival under oxidative stress. Biological assays validated HpTpx inhibition as the origin of the activity boost, demonstrated by covalent modification of the catalytic cysteine and co-crystallization. Metro-P3 and refined ether analogs exhibited favorable pharmacological profiles with no cytotoxicity. The activity boost translated to an in vivo H. pylori mouse model demonstrating full eradication at 50-fold reduced dosing compared to metronidazole triple therapy. Our results highlight 5-nitroimidazole ethers as dual mode of action antibiotics which induce oxidative stress and inhibit the stress response leading to bacterial death.","dates":{"publication":"2026-04-13","submission":"2025-08-18"},"accession":"PXD067473","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:2042546","NEWT:259447","NEWT:295546","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:1584","NEWT:36630","NEWT:383379","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:7719","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:2065263","NEWT:177437","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:485","NEWT:56636","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:237610","NEWT:95486","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:906914","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:550","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:1613","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:298176","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:40674","NEWT:1194669","NEWT:443906","NEWT:13037","NEWT:519","NEWT:2510939","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:105487","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:300641","NEWT:39655","NEWT:9554","NEWT:475932","NEWT:256318","NEWT:206411","NCBITaxon:6191","NEWT:229533","NEWT:214053","NEWT:28550","NEWT:80863","NEWT:90675","NEWT:52639","NEWT:52638","NEWT:57075","NEWT:1666905","NEWT:8697","NEWT:861109","NEWT:8695","NEWT:884204","NEWT:1045775","NEWT:1123869","NEWT:9544","NEWT: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