{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/PrideUpload_ACSCentralSci_Fasan.xlsx"],"Msf":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_17-163.msf"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/checksum.txt"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_Ctrl-2Black_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_MCL-4Blue_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_Ctrl-4Blue_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_Ctrl-2Blue_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_MCL-2Black_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_Ctrl-14Blue_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_MCL-2Blue_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_MCL-14Blue_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_Ctrl-4Black_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_Ctrl-14Black_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_MCL-14Black_17-163.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD024455/Alwaseem_MCL-4Black_17-163.raw"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["rudi.fasan@rochester.edu"],"submitter":["Kevin Welle"],"technology_type":["Affinity purification coupled with mass spectrometry proteomics","Mass Spectrometry"],"software":[""],"submitter_keywords":["Biocatalysis","Sesquiterpene lactones","Leukemia stem cells","Multi-probe proteomics","C-h functionalization","Competitive pull-down"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD024455"],"sample_protocol":["M9-ENL1 cells were harvested by centrifugation (500 x g, 10 min) and washed 5x with cold phosphate buffered saline to remove the culture medium. The cell pellet was lysed in cold RIPA buffer containing a protease inhibitor (pi) cocktail for 10 minutes at 4 ° C followed by sonication. The lysate was centrifuged (10,000 g, 10 min) to remove cell debris and the protein concentration was determined using the BCA reagent per manufacturer’s instruction (ThermoFisher). M9-ENL1 cell lysates (2 mg protein per sample) were incubated with 10 μM MCL (MCL-treated control) or just DMSO (sample) for 1 hour on ice with gentle shaking. The samples were then treated with the appropriate biotinylated MCL-based probe at a final concentration of 10µM (control and non-control groups). The lysate was incubated on ice for 2 hours with gentle shaking and transferred to a 3 kDa centrifugal filter unit. The samples were washed with cold PBS (+ pi) three times to remove any unreacted probe and incubated with 50 µl of ultra-high capacity neutravidin beads (100 µl of slurry, Pierce) for 60 minutes at 4 ° C with gentle shaking. Cold phosphate buffer saline (PBS) (+ pi) was added to the mixture and the samples were centrifuged for 2 min at 10,000 rpm (2x). Beads were successively washed with 1 mL of PBS containing pi and 2% SDS and the supernatant was removed via centrifugation (2 min, 10000 rpm, 4X). 40 μL of 1× laemli sample buffer was added to the beads and then beads were heated at 95 ° C for 10 minutes to elute the neutravidin bound proteins. The pull-down experiments were performed in triplicate for each probe.  Samples were run into a 4-12% SDS-PAGE gel to create a ~10 mm length region, allowing total protein to be evaluated in a single gel digest.  After staining with SimplyBlue SafeStain (Invitrogen), these regions were excised, cut into 1 mm cubes, de-stained, then reduced and alkylated with DTT and IAA, respectively (Sigma).  Gel pieces were dehydrated with acetonitrile.  Aliquots of trypsin (Promega) were reconstituted to 10 ng/µL in 50 mM ammonium bicarbonate and added so that the solution was just covering the dehydrated gel pieces.  After half an hour at room temp, additional ammonium bicarbonate was added until the gel pieces were completely submerged and placed at 37 °C overnight.  Peptides were extracted the next day by addition of 50% acetonitrile, 0.1% TFA, then dried down in a CentriVap concentrator (Labconco).  Peptides were desalted with homemade C18 spin columns, dried again, and reconstituted in 0.1% TFA. Peptides were injected onto a homemade 30 cm C18 column with 1.8 um beads (Sepax), with an Easy nLC-1000 HPLC (Thermo Fisher), connected to a Q Exactive Plus mass spectrometer (Thermo Fisher).  Solvent A: 0.1% formic acid in water, Solvent B : 0.1% formic acid in acetonitrile.  Gradient: 3% B for 2 minutes, then ramp to 30% B over 41 minutes, then to 70% over 3 minutes, then 70% for 4 minutes, then re-equilibrated to 3%, for a total run time of 60 minutes.  The Q Exactive Plus was operated in data-dependent mode, with a full MS1 scan followed by 10 data-dependent MS2 scans.  The full scan was done over a range of 400-1400 m/z, with a resolution of 70,000 at m/z of 200, an AGC target of 1e6, and a maximum injection time of 50 ms.  The MS2 scans were performed at 17,500 resolution, with an AGC target of 5e4 and a maximum injection time of 120 ms.  The isolation width was 1.5 m/z, with an offset of 0.3 m/z, and a normalized collision energy of 27."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Raw data was searched using the SEQUEST search engine within the Proteome Discoverer software platform, version 2.2 (Thermo Fisher), using the SwissProt human database.  Trypsin was selected as the enzyme allowing up to 2 missed cleavages, with an MS1 mass tolerance of 10 ppm, and an MS2 mass tolerance of 25 mmu.  Carbamidomethyl was set as a fixed modification, while oxidation of methionine was set as a variable modification.  Minora was used to determine relative protein abundance between samples. Normalization was performed using the Total Peptide Amount option in the Precursor Ions Quantifier Node. Percolator was used as the FDR calculator, filtering out peptides which had a q-value greater than 0.01. Missing values were imputed from the normal distribution using Perseus software. The identified proteins were evaluated using an unpaired t-test between the MCL treated (control) and DMSO treated (sample) groups with a p-value cutoff of 0.05."],"omics_type":["Proteomics"],"labhead":["Rudi Fasan"],"instrument_platform":[""],"labhead_affiliation":["Department of Chemistry, University of Rochester, United States"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"publication":["34079900 Alwaseem H, Giovani S, Crotti M, Welle K, Jordan CT, Ghaemmaghami S, Fasan R. Comprehensive Structure-Activity Profiling of Micheliolide and its Targeted Proteome in Leukemia Cells via Probe-Guided Late-Stage C-H Functionalization. ACS Cent Sci. 2021 7(5):841-857 10.1021/acscentsci.0c01624"],"submitter_mail":["kevin_welle@urmc.rochester.edu"],"submitter_affiliation":["University of Rochester"],"submitter_country":["United States"],"pubmed_abstract":["The plant-derived sesquiterpene lactone micheliolide was recently found to possess promising antileukemic activity, including the ability to target and kill leukemia stem cells. Efforts toward improving the biological activity of micheliolide and investigating its mechanism of action have been hindered by the paucity of preexisting functional groups amenable for late-stage derivatization of this molecule. Here, we report the implementation of a probe-based P450 fingerprinting strategy to rapidly evolve engineered P450 catalysts useful for the regio- and stereoselective hydroxylation of micheliolide at two previously inaccessible aliphatic positions in this complex natural product. Via P450-mediated chemoenzymatic synthesis, a broad panel of novel micheliolide analogs could thus be obtained to gain structure-activity insights into the effect of C2, C4, and C14 substitutions on the antileukemic activity of micheliolide, ultimately leading to the discovery of \"micheliologs\" with improved potency against acute myelogenic leukemia cells. These late-stage C-H functionalization routes could be further leveraged to generate a panel of affinity probes for conducting a comprehensive analysis of the protein targeting profile of micheliolide in leukemia cells via chemical proteomics analyses. These studies introduce new micheliolide-based antileukemic agents and shed new light onto the biomolecular targets and mechanism of action of micheliolide in leukemia cells. More broadly, this work showcases the value of the present P450-mediated C-H functionalization strategy for streamlining the late-stage diversification and elucidation of the biomolecular targets of a complex bioactive molecule."],"pubmed_title":["Comprehensive Structure-Activity Profiling of Micheliolide and its Targeted Proteome in Leukemia Cells via Probe-Guided Late-Stage C-H Functionalization."],"pubmed_authors":["Alwaseem Hanan H, Giovani Simone S, Crotti Michele M, Welle Kevin K, Jordan Craig T CT, Ghaemmaghami Sina S, Fasan Rudi R"],"sample_synonyms":["mantle cell, Methane, sodium salt, Water, ammonium formate, 13C-labeled, IL1BC, MeCN, cadmium salt, TFA, CASP-1, H(2)O, Physical, DCAF2, Ramp, BOUND WATER, chronic obstructive pulmonary disease (COPD), SDH, 2-(indol-3-yl)ethanoic acid, magnesium formate, oxidane, zinc salt, Solvent, prevention, Long Term, thomson., WATER, l(2)k09905, HOH, MYM, Bca, l(1)AA33, dmTAF[[II]]230, SDS, Polypeptides, ramp, T22F8_160, cobalt(II) formate dihydrate, Protease Inhibitor, ATGPR7, CH3-C#N, B1, High Performance Liquid Chromatography, Chronic irreversible airway obstruction, 1, DGS, GRP8, 2, GRP7, AGM4, RAMP, Il1bc, 5730564G15Rik, bca, DmelCG8428, tetraoxophosphate(V), LRCC, Spin, Polyacrylamide, TFIID TAF250, SDS-PAGE electrophoresis, cel, tetraoxidophosphate(3-), dimethyl sulphoxide, CAFL - Chronic airflow limitation, T6G21.3, IL-1BC, HPLC, AI047805, 2810047L02Rik, geographical area, SPIN, Peptidohydrolase Inhibitor, dehydrated, h, l(1)B2/13.1, disease (COPD), BTKAP1, sample, D1, Chromatography, CT, High Speed Liquid, GTFII-I, Inorganic, Dimethylsulfoxid, Histological Labeling, nickel salt, 6030441I21Rik, Physical Barrier, sulfinylbis-, chronic obstructive lung disease [Ambiguous], dTAF[[II]]230, DMSO, preventive therapy, l(1)LB9, Stars, Caspase-1 subunit p10, BZRP, dmso, cobalt (+2) salt, Ct, Liquid Chromatography, TAF200, Longterm Effect, BAP135, DmelCG7788, PBS, PBR, CHRONIC OBSTRUCTIVE AIRWAY DIS, (CH3)2SO, H2O, (Indol-3-yl)acetate, sodium (4:1:1) salt, Dm1, magnesium salt, Hydrolase Inhibitor, HLRCC, BAP-135, Airflow Obstruction, Sulfinylbis(methane), Hydrogen Oxide, GLYCINE-RICH PROTEIN 8, Chronic Obstructive Airways Disease, Chronic obstructive lung disease, Antagonists, Sodium Dodecyl Sulfate-PAGE, Barrier, LY57, Rimso50, formate, Shwachman-Diamond type metaphyseal dysplasia, reagent, cromium (+3), Shwachman syndrome, GtfII-I, beta Trypsin, l2dtl, CG1106, Speed, lymphoma, Painful bladder syndrome, heteroauxin, l(2)W5, F2G1.4, Taf250, OBSTRUCTIVE PULMONARY DISEASE (COPD), Indicator, Drice, ammonium (4:1) salt, Endopeptidase, Peptide Peptidohydrolase Inhibitor, methyl cyanide, Sodium Dodecyl Sulfate-PAGEs, DRICE, lead (+2) salt, STARS, TAF230, Dimethyl, BPBS, F10B6_15, tetraoxophosphate(3-), dtl-b, l(1)VE614, nickel formate dihydrate, Access, lead formate, region or site annotation, Effects, Endopeptidase Inhibitors, DrIce, DrICE, aluminum salt, CAO - Chronic airflow obstruction, Pierce, dimethylsulfoxyde, Gel Electrophoresis, CHRONIC OBSTRUCTIVE PULM DIS, Chronic obstructive pulmonary disease NOS, protein-containing complex, abdominal muscles, SDS polyacrylamide gel electrophoresis of proteins, thomson, Chalk, CG7826, and RNA binding 1, F10B6.15, agua, mass-to-charge ratio, Chronic obstructive pulmonary disease finding, culture filtrate, Sulphoxide, PULMONARY DISEASE (COPD), Ly57, Polyacrylamide Gel Electrophoresis, CG7835, Gene Products, CG42273, Peptide Hydrolase, sulfinylbis(methane), positional, dTAF[[II]]250, thallium (+1) salt, Longterm, cell, PCE-2, beta-Trypsin, rubidium salt, 4733401P19Rik, pk18, Long-Term, methanoic acid, Antagonist, kf, MBR, High-Performance Liquid Chromatographies, Solution, CD156, Rimso-50, Injectable, dTAF250, dimetil sulfoxido, Rimso, ATGRP7, Protease Antagonists, Caspase-1 subunit p20, ATGRP8, congenital lipomatosis of pancreas, NEC, PTBR, Chronic, Peptide Peptidohydrolase Inhibitors, peptidos, UPLC, MCL, PTHB1, LB5, SDS PAGE, [PO4](3-), WBSCR6, 1-(14)C-labeled, chronic obstructive airways disease NOS, caspase 3, COAD, Proteins, Architectural Barrier, Facility Accesses, Architectural Barriers, BG:DS00004.13, Stainings, potassium formate, CE-2, Mischung, copper (+2) salt, Rnt, COLD - 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Temecula1, Sonications, P45, L2DTL, Control, cobaltous formate, hydrogen hydroxide, cdt2-b, SDS polyacrylamide gel electrophoresis, cdt2-a, Accesses, AW124434, CG17603, TAF[[II]], acqua, Chronic Airflow Obstruction, Peptidase Inhibitors, Labelings, phosphate, WBS, phosphate ions, COPD - Chronic obstructive pulmonary disease, SR3-5, copper salt, chronic obstructive airways disease, Antiproteases, p45, CG8428, Liquid, Polypeptide, Wasser, obstructive pulmonary disease (COPD), Inhibitor, COLD, chronic obstructive lung disease, Bladder Pain Syndrome, CG1849, d230, DISEASE (COPD), Reagent, Histological Labelings, cesium salt, Gene, Protease, dTAFII250, Rimso 100, circadian rhythm, EfW1, TFII-I, Buffer, Bruton tyrosine kinase-associated protein 135, formic acid, Cold, Sodium Dodecyl Sulfate PAGE, potassium salt, Pulmonary Disease, reduced, dmTAF1, Ms1, Taf230, Injection, 14C-labeled, Tina, Histological, IB291, Protease Antagonist, tiny, AA673430, Reagents and Indicators, Pro-Mega, TAF250, S(O)Me2, Cold Temperatures, Taf200, Glass, Staining, reactivo, MS1, MS2, Runt, gel, Inorganic Phosphates, Taf1p, Indoleacetic acid, eau, calcium formate, Chronic Obstructive, Indicators, Physical Barriers, Xylella fastidiosa (strain Temecula1 / ATCC 700964), 5830413P05Rik, lysate, Dimethylsulphinyl, DmelCG11387, (indol-3-yl)acetic acid, male sterility 1, Control Group, Ultra Performance Liquid Chromatography, cromium (+3) salt, Long-Term Effect, AA553322, BTK-associated protein 135, TAF, MCUL1, Facility, sodium formate, BLNK-S, Controlled, COPD, High-Performance Liquid Chromatography, small, DYRK1, COAD - Chronic obstructive airways disease, Controlling, 5E4, TAF[[II]]250, culture medium, nickel formate, DBI, 3H-labeled, protein complex, not elsewhere classified, DmelCG1849, strontium formate, l(3)84Ab, strontium salt, Labeling, PULM DIS CHRONIC OBSTRUCTIVE, Peptide, Chronic obstructive pulmonary disease finding (finding), PKBS, Dimexide, p230, water, Protein, sequence, TFIID, Dyrk1, 25/11, GLYCINE-RICH RNA-BINDING PROTEIN 7, CES2A1, Sulfoxide, ACETONITRILE, tandem MS, cyanomethane, chronic obstructive airway disease, ZNF198, Temperature, Architectural, Accessibility, with urinary tract Abnormality and cryptorchidism, TAF[[II]]230, PRSS, MALE STERILITY 1 PROTEIN, Dops, CC1, Tripcellim, TAF[II]250, l(1)19Ea, CGI-97, sample population, chromic formate, Protein Gene Products, Interleukin-1 beta convertase, l(2)10403, P235, Trypure, DmelCG17603, concentration, FIM, 3.4.22.36, calcium salt, cold (chronic obstructive lung disease), TEMP, Peptid, High Performance Liquid, Chronic Obstructive Lung Disease, Barriers, SCLL, dimethyl sulfur oxide, TAF1"],"name_synonyms":["leukemia, Leucocythemia, Activity, conformation, leukaemia NOS, total expressed protein, Cell., Leucocythemias, Leucocythaemia, leukaemia, General activity, Proteomes, Leukemias, Leucocythaemias"],"data_synonyms":["mantle cell, Methane, IPP2A2, ion, Dm NinaC, NINA C, CG5125, protein, prevention, Normal Distributions, 5730420M11Rik, peptide, Polypeptides, L-Isomer Methionine, Methionine, peptido, dorsal marginal zone, Software Engineering, protein aggregate, prevention and control, Computer Program, WMS, Rheumabene, SET, LRCC, Man (Taxonomy), peptides, reference sample, TAF-I, M, Open, E430039A18Rik, dimethyl sulphoxide, Computer Programs and Programming, T6G21.3, DmelCG4299, preventive measures, IGAAD, set, Nina C, Dimethylsulfoxide, DmelCG10574, ppm, Normal, GPHYSD2, Racemethionine, Dimethylsulfoxid, CT16120, SGS, phapii, sulfinylbis-, DMSO, Stars, preventive therapy, millidalton, dimethyli sulfoxidum, dmso, Modern, methylsulfinylmethane, 2-Amino-4-(methylthio)butyric acid, StF-IT-1, Search, Source Softwares, Software Tools, Programs, DmelCG5125, ACMICD, Program, Computer Applications, (CH3)2SO, Ionen, Computer Applications Software, Distributions, Computer Applications Softwares, Henson's node, Methionin, HLRCC, Softwares, Hmet, Sulfinylbis(methane), DRONINAC, Rimso 50, parent ion, Software Applications, HLA-DR-associated protein II, DI-2, FDR adjusted p-value, MS/MS, Rimso50, Source Software, I-2Dm, mmu, MASS, CG4299, beta Trypsin, human, I-2PP1, sample., Applications, TAF-IBETA, lymphoma, precursor ion, Biocatalyst, DMZ, TAF-Ibeta, Polypeptide, STARS, i2pp2a, Liquimeth, Gaussian, Computer Software Applications, Dimethyl, human being, 2-amino-4-(methylsulfanyl)butanoic acid, Biocatalysts, L Isomer, FBN, Gene, dimethylsulfoxyde, Rimso 100, Computer, precursor, protein-containing complex, PHAPII, Human, Sulphoxide, Gaussian Distribution, Homo sapiens, Ms1, ECTOL1, Gene Products, L-Methionine, Distribution, NINAC, Man, Pedameth, Application, CG54125, S(O)Me2, Open Source Softwares, sulfinylbis(methane), Search Engines, NinaC, ninac, MS1, nodus primitivus, Software Application, MS2, iones, ipp2a2, beta-Trypsin, Open Source Software, 2pp2a, ions, CG10574, CG 5125, CD156, Computer Software Application, OCTD, Rimso-50, dimetil sulfoxido, Enzyme, Rimso, Dimethylsulphinyl, 2PP2A, 2-amino-4-(methylthio)butanoic acid, Tools, 10^[-6], taf-ibeta, stem node, male sterility 1, dSET, dSet, peptidos, 2.2, MCL, MCUL1, metionina, Controlled, Applications Software, Open Source, Controlling, Computer Software, milli unified atomic mass unit, DL-Methionine, protein complex, Proteins, igaad, L-Isomer, Peptide, Engine, Tool, group, polypeptide, Software Tool, native protein, I-2PP2A, Dimexide, Dimethylsulphoxide, Dm I-2, Protein, I2PP2A, CT42491, MFS1, Sulfoxide, Software, tandem MS, Data Base, WMS2, Met, CLECSF8, PRSS, ensemble, nodal stem, MALE STERILITY 1 PROTEIN, mDa, prophylaxis, Engineering, Striated muscle activator of Rho-dependent signaling, Tripcellim, dimethyl sulfoxide, Sclerosol, Dimethyl Sulphoxide, CD156a, sample population, Protein Gene Products, Computer Programs, Trypure, Gene Proteins, dSET/TAF-Ibeta, 2610030F17Rik, Applications Softwares, Ion, control, Modern Man, SSKS, alpha-amino-gamma-methylmercaptobutyric acid, Peptid, dimethyl sulfur oxide, AA407739"],"description_synonyms":["9b1, Activity, 9b2, P450-A, determination, CYP9C1, P450-B, protein sorting along secretory pathway, Pflanze, Eig17-1, Visible Light, Progress Reports, viridiplantae, 9c1, DmelCG6816, Summary Report, CYP9B1, Mother Cells, 91R, Summary Reports, multicellular organismal biosynthetic process, 18a1, CYPIIB6, single-organism biosynthetic process, Progress Report, DmelCG3656, cyp12a4, CYP2B7, NCPR, Leucocythemias, BF6-2, T1, CytP450, 143127_at, cyp6a2, Progress, TNFSF14, Field Reports, 6a20, CG3972, HAI, 6a22, CG1438, CG3616, CYP2B7P, Radiation, wide/broad, UNQ391/PRO726, CYP6A2, DmelCG10240, P450-B1, CYP6A8, CYP6A9, DmelCG11567, Light, CG6816, Acute onset, 6a2, Cyt-P450-rBF6-2, LIGHT, Colony-Forming Unit, Stem Cell, Investigative Report, CYP6a2, 6a8, P-450, 6a9, ESTS:181H6T, Hydroxylations, viral haemagglutinin inhibition assay, Mother Cell, Visible Radiations, ASC-T1, cyp9b2, P-450 Cyp9b2, Leucocythemia, Visible Radiation, 91-R, DmelCG3972, HVEML, DmelCG1438, Stem, DmelCG3616, Field, Plant, Colony Forming Units, Cyt-P450-B1, CG3656., Cyp6A2, P450A, wide, Report, DMR, Cyt-P450-17C, Cyp6a, Cells, DmelCG10248, DmelCG10245, DmelCG10246, CG3656, DmelCG4486, DmelCG4485, conformation, Colony-Forming Units, CG3466, Progenitor Cell, CYP12A4, BEST:GH08116, broad, anon-WO0140519.102, Ly113, EG:152A3.4, hydroxylation, Investigative, cyp4g1, CYP6D1, leukaemia NOS, Cyt-P450-rAF5, Cyt-P450-rAF4, 181H6T, Leucocythaemia, Cyt-P450-A1, EFVM, leukaemia, Progenitor, plantae, hemagglutination inhibition, CYP18A1-Dm, EG:87B1.1, CG6042, cyp4d1, Mother, Visible, P450, HIA, scgamma, p450, IIB1, CG9438, TR2, Investigative Reports, CPB6, leukemia, CG11567, 4d1, 4d2, CPR, 4C3, Colony Forming Unit, synthesize, Progenitor Cells, Cyt-P450-4D1, Cyt-P450-4D2, CYP18, CD258, Leukemias, Cell, cpr, cyp4c3, CYP4D1, DmelCG3466, CYP4D2, nascent polypeptide association, Cyp18, Research Reports, chemical analysis, CYP2B, CG4485, P-450-A, P-450-B, CG4486, Leucocythaemias, DmelCG9438, Radiations, CT20826, biological activity, 12a4, EG:165H7.1, cyp18, DmCPR, HVEM-L, Photoradiation, cyp18a1, 4g1, LTg, DmelCG6042, CG10248, CG10245, cyp6a22, CG10246, Photoradiations, Reports, CCR, assay, Cyp4D1, Summary, CG10240, General activity, Field Report"],"pubmed_abstract_synonyms":["mode of action, pharmacodynamics, 9b1, Activity, 9b2, Product, P450-A, determination, CYP9C1, P450-B, protein sorting along secretory pathway, Pflanze, Eig17-1, Visible Light, Progress Reports, viridiplantae, 9c1, DmelCG6816, Biological, Summary Report, CYP9B1, Mother Cells, Biological Product, 91R, Summary Reports, multicellular organismal biosynthetic process, 18a1, CYPIIB6, single-organism biosynthetic process, Biologic Drugs, Progress Report, DmelCG3656, cyp12a4, Natural, CYP2B7, NCPR, Leucocythemias, BF6-2, Biological Drugs, T1, CytP450, 143127_at, cyp6a2, Progress, TNFSF14, Biological Medicine, Field Reports, 6a20, CG3972, Medicine, HAI, 6a22, stage, Medicines, CG1438, CG3616, Biologic Drug, CYP2B7P, Radiation, Biologic Products, UNQ391/PRO726, CYP6A2, DmelCG10240, Aptitudes, P450-B1, CYP6A8, CYP6A9, DmelCG11567, Light, CG6816, Ability, Acute onset, 6a2, Cyt-P450-rBF6-2, LIGHT, Colony-Forming Unit, Stem Cell, Investigative Report, Biopharmaceuticals, CYP6a2, 6a8, P-450, 6a9, Biologic Product, ESTS:181H6T, Hydroxylations, viral haemagglutinin inhibition assay, Mother Cell, Visible Radiations, ASC-T1, cyp9b2, P-450 Cyp9b2, Leucocythemia, Visible Radiation, 91-R, DmelCG3972, HVEML, Biological Medicines, DmelCG1438, Stem, DmelCG3616, Biological Drug, Field, Plant, Colony Forming Units, Cyt-P450-B1, Biologics, Cyp6A2, P450A, Report, DMR, Cyt-P450-17C, Cyp6a, Cells, DmelCG10248, DmelCG10245, DmelCG10246, developmental stage., CG3656, DmelCG4486, DmelCG4485, Biologic Pharmaceuticals, conformation, Peptidomics, Colony-Forming Units, developmental stage, CG3466, Progenitor Cell, CYP12A4, BEST:GH08116, anon-WO0140519.102, Ly113, EG:152A3.4, hydroxylation, Investigative, cyp4g1, CYP6D1, leukaemia NOS, Cyt-P450-rAF5, Cyt-P450-rAF4, 181H6T, Leucocythaemia, Cyt-P450-A1, EFVM, leukaemia, Progenitor, Biologicals, plantae, Drugs, hemagglutination inhibition, CYP18A1-Dm, EG:87B1.1, CG6042, cyp4d1, Natural Product, Mother, Visible, P450, HIA, scgamma, Abilities, mechanism of action, p450, IIB1, CG9438, TR2, Investigative Reports, Biologic, CPB6, Pharmaceuticals, leukemia, CG11567, 4d1, 4d2, Products, CPR, 4C3, Colony Forming Unit, Biologic Medicines, synthesize, Progenitor Cells, Cyt-P450-4D1, Cyt-P450-4D2, CYP18, CD258, Leukemias, Cell, cpr, cyp4c3, CYP4D1, DmelCG3466, CYP4D2, nascent polypeptide association, Cyp18, Research Reports, chemical analysis, Biopharmaceutical, CYP2B, CG4485, P-450-A, P-450-B, CG4486, Leucocythaemias, DmelCG9438, Radiations, CT20826, biological activity, 12a4, EG:165H7.1, cyp18, DmCPR, HVEM-L, Photoradiation, Talents, cyp18a1, 4g1, LTg, DmelCG6042, Drug, CG10248, CG10245, cyp6a22, CG10246, Photoradiations, pharmacologic action, Reports, Talent, Natural Products, CCR, assay, Cyp4D1, Summary, CG10240, General activity, Field Report"],"pubmed_title_synonyms":["leukemia, Leucocythemia, Activity, conformation, leukaemia NOS, total expressed protein, Leucocythemias, stage, Leucocythaemia, leukaemia, General activity, Proteomes, developmental stage., Leukemias, Cell, Leucocythaemias"],"additional_accession":[]},"is_claimable":false,"name":"Comprehensive Structure-Activity Profiling of Micheliolide and its Targeted Proteome in Leukemia Cells via Probe-Guided Late-Stage C—H Functionalization","description":"The plant-derived sesquiterpene lactone micheliolide was recently found to possess promising antileukemic activity, including the ability to target and kill leukemia stem cells. Efforts toward improving the biological activity of micheliolide and investigating its mechanism of action have been hindered by the paucity of preexisting functional groups amenable for late-stage derivatization of this molecule. Here, we report the implementation of a probe-based P450 fingerprinting strategy to rapidly evolve engineered P450 catalysts useful for the regio- and stereoselective hydroxylation of micheliolide at two previously inaccessible aliphatic positions in this complex natural product. Via P450-mediated chemoenzymatic synthesis, a broad panel of novel micheliolide analogs could thus be obtained to gain structure-activity insights into the effect of C2, C4, and C14 substitutions on the anti-leukemic activity of micheliolide, ultimately leading to the discovery of ‘micheliologs’ with improved potency against acute myelogenic leukemia cells. These late-stage C—H functionalization routes could be further leveraged to generate a panel of affinity probes for conducting a comprehensive analysis of the protein targeting profile of micheliolide in leukemia cells via pull-down experiments and proteomic analyses. These studies introduce new micheliolide-based anti-leukemic agents and shed light onto the biomolecular targets and mechanism of action of micheliolide in leukemia cells. More broadly, this work showcases the value of the present P450-mediated C—H functionalization strategy for streamlining late-stage diversification and elucidation of the biomolecular targets of a complex bioactive natural product.","dates":{"publication":"2021-09-10","submission":"2021-03-02"},"accession":"PXD024455","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:184922","NEWT:3555","NEWT:2","NEWT:157546","NEWT:35554","NEWT:38942","NEWT:307972","NEWT:32046","NEWT:544496","NEWT:2042546","NEWT:45351","NEWT:43179","NEWT:4513","NEWT:5722","NEWT:376741","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:309800","NEWT:1211601","NEWT:876138","NEWT:3654","NEWT:237561","NEWT:5833","NEWT:6928","NEWT:10036","NEWT:36745","NEWT:1351","NEWT:1438992","NEWT:2649997","NEWT:272563","NEWT:224326","NCBITaxon:79857","NEWT:1096976","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:1589","NEWT:135622","NCBITaxon:4896","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:3880","NEWT:272559","NEWT:3641","NEWT:383379","NEWT:466585","NEWT:10029","NEWT:913645","NEWT:1000589","NEWT:85963","NEWT:85962","NEWT:317447","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:398580","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:84645","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:5888","NEWT:1283","NEWT:931281","NEWT:4550","NEWT:1000561","NEWT:197","NEWT:1390363","NEWT:288705","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:44689","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:1270","NEWT:374990","NEWT:2242","NEWT:4784","NEWT:11320","NEWT:360106","NEWT:286","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:44685","NEWT:317513","NEWT:1148","NEWT:580240","NEWT:294128","NEWT:11676","NEWT:55571","NEWT:100226","NEWT:4530","NEWT:4896","NEWT:75058","NEWT:13616","NEWT:1390","NEWT:1094343","NEWT:296543","NEWT:1773","NEWT:1895","NEWT:1182590","NEWT:3712","NEWT:105023","NEWT:935293","NEWT:64152","NEWT:4924","NEWT:749200","NEWT:375146","NEWT:990346","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:263","NEWT:230741","NEWT:52283","NEWT:284812","NCBITaxon:1313","NEWT:43330","NEWT:1603293","NEWT:408169","NEWT:44544","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:243277","NEWT:990119","NEWT:408172","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:257313","NEWT:400772","NEWT:3708","NEWT:128161","NEWT:332648","NEWT:106592","NEWT:536231","NEWT:460519","NEWT:1187947","NEWT:1432138","NEWT:10312","NEWT:1424507","NCBITaxon:1773","NEWT:9598","NEWT:8030","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:67767","NEWT:432359","NEWT:46835","NEWT:1182263","NEWT:2711","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:29159","NEWT:253","NEWT:10306","NCBITaxon:2759","NEWT:1233435","NEWT:93061","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:240906","NEWT:593117","NEWT:3635","NEWT:5811","NEWT:235443","NEWT:272623","NEWT:272624","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:169963","NEWT:32644","NEWT:225117","NEWT:499175","NEWT:109779","NEWT:476272","NEWT:3747","NEWT:195051","NEWT:367830","NEWT:1255228","NEWT:178616","NEWT:410289","NEWT:373153","NEWT:352472","NEWT:357","NEWT:360094","NEWT:470","NEWT:1313","NEWT:411469","NEWT:84023","NEWT:559292","NEWT:39491","NCBITaxon:5811","NEWT:411464","NEWT:411460","NEWT:2014887","NEWT:2762","NEWT:1174673","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:571256","NEWT:28038","NEWT:1663","NEWT:1423","NEWT:4932","NEWT:3603","NEWT:2759","NEWT:3847","NEWT:327159","NEWT:178876","NEWT:327160","NEWT:573","NEWT:9031","NEWT:7091","NEWT:108931","NEWT:241368","NEWT:42528","NEWT:190802","NEWT:9778","NEWT:150475","NEWT:303","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216979","NEWT:5180","NEWT:256737","NEWT:115104","NEWT:1121114","NEWT:663","NEWT:1081927","NEWT:1238993","NEWT:67825","NEWT:185579","NEWT:941442","NEWT:220668","NEWT:13076","NEWT:1249668","NEWT:7108","NEWT:317","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:9402","NEWT:415540","NEWT:550","NEWT:675060","NEWT:4081","NEWT:334542","NEWT:554","NEWT:98334","NEWT:426428","NEWT:7574","NEWT:1715256","NEWT:7215","NEWT:575412","NEWT:29204","NEWT:2172103","NEWT:507601","NEWT:643680","NCBITaxon:6157","NEWT:746360","NEWT:6239","NEWT:470150","NEWT:216257","NEWT:102169","NEWT:9986","NEWT:4054","NEWT:73239","NEWT:226186","NEWT:1268063","NEWT:8782","NEWT:1263854","NEWT:435590","NEWT:1902","NEWT:160488","NEWT:28104","NEWT:1908","NEWT:13164","NEWT:216129","NCBITaxon:2","NEWT:985076","NEWT:1215323","NEWT:52641","NEWT:7038","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:353152","NEWT:2829","NEWT:366581","NEWT:216599","NEWT:216595","NEWT:1194669","NEWT:51329","NEWT:243230","NEWT:8355","NEWT:9685","NEWT:7029","NEWT:1080772","NEWT:8479","NEWT:1283300","NEWT:6183","NEWT:6063","NEWT:630","NEWT:334747","NEWT:61235","NEWT:15368","NEWT:6289","NEWT:436486","NEWT:6287","NEWT:300641","NEWT:727","NEWT:9796","NEWT:725","NEWT:170187","NEWT:469008","NEWT:260707","NEWT:256318","NCBITaxon:6191","NEWT:1836","NEWT:185431","NEWT:29760","NEWT:260704","NEWT:703612","NEWT:260705","NEWT:80863","NEWT:2697049","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