CYP82C2 has been recruited into the WRKY33 regulon and indole-3-carbonylnitrile (ICN) biosynthetic pathway through exaptation of a retroduplicated LINE retrotransposon (EPCOT3) into an enhancer. The stepwise development of a chromatin-accessible WRKY33-binding site on EPCOT3 has potentiated the regulatory neofunctionalization of CYP82C2 and the evolution of inducible defense metabolite 4-hydroxy-ICN in Arabidopsis thaliana. Although transposable elements (TEs) have long been recognized to have the potential to rewire regulatory networks, these results establish a more complete understanding of how duplicated genes and TEs contribute in concert to chemical diversity and pathogen defense.Plants synthesize numerous ecologically specialized, lineage-specific metabolites through biosynthetic gene duplication and functional specialization. However, it remains unclear how duplicated genes are wired into existing regulatory networks. We show that the duplicated gene  Arabidopsis plants can produce 4-hydroxyindole-3-carbonitrile (4OH-ICN) upon pathogen infection. Here, the authors show that EPCOT3, a retrotransposonderived enhancer, mediates WRKY33-binding, pathogen-responsive transcription of CYP82C2, and synthesis of 4OH-ICN. Plant-specialized metabolic enzymes are ultimately produced from primary metabolic enzymes through gene duplication and subsequent functional divergence of one or both paralogs to produce enzymes with altered expression patterns and/or protein functions5. They are also often organized into transcription factor (TF) regulons of co-regulated genes for optimal timing, amplitude, and tissue-specific pathway gene expression and subsequent metabolite accumulation7.Plant secondary or specialized metabolites are essential for plant survival in co-evolving biotic and fluctuating abiotic environments. The evolutionary process of chemical innovation resulted in the collective synthesis of hundreds of thousands of ecologically specialized, mostly lineage-specific metabolitescis-regulatory modules such as enhancers and promoters can accelerate the capture of duplicated genes into regulons, thus driving phenotypic diversity10. Enhancers consist of TF binding sites (TFBSs) and are derived either through mutation or co-option of a TFBS-carrying transposable element (TE)11. TE exaptations are hypothesized to be responsible for the rapid transcriptional rewiring of gene regulatory networks in ancient lineages of vertebrates14 and plants15, but general understandings of the physiological significance of this rewiring are greatly limited.Changes in 16. Pathogenic bacteria additionally compromise PTI via specific virulence effector proteins 16. PTI involves the extracellular perception of conserved molecules known as microbe-associated molecular patterns (MAMPs), whereas ETI involves the cytosolic perception of effectors. Although ETI results in the formation of more rapid and robust pathogen-specific responses including a form of programmed cell death known as the hypersensitive response (HR)16, both result in the ability of naive host cells to generate, through non-self perception and subsequent transcriptional reprogramming, pathogen-inducible specialized metabolites necessary for defense19.Bacteria elicit two primary immune defense modes in plants, pattern- and effector-triggered immunity (PTI and ETI)20, camalexin22, and 4-hydroxyindole-3-carbonylnitrile (4OH-ICN)23—are known to expand innate immunity in Arabidopsis thaliana. Their biosynthetic pathways share an early step, which is the conversion of Trp to indole-3-acetaldoxime (IAOx) via the genetically redundant P450 monooxygenases CYP79B2 and CYP79B326 , whereas camalexin is restricted to the Camelineae tribe of Brassicaceae30. The evolutionary conservation of 4OH-ICN has not yet been investigated.Three pathogen-inducible tryptophan (Trp)-derived defense metabolites— 4-methoxyindol-3-ylmethylglucosinolate (4M-I3M)326 Fig. . The cam 28 Fig. . CYP71A1ICN Fig. 23. 4M-I3A. thaliana32 and its orthologs regulate numerous unrelated specialized metabolites in other flowering plant lineages33. The group I class of WRKYs to which WRKY33 belongs is an ancient clade of regulators; orthologs in the green alga Chlamydomonas reinhardtii may be ancestral to all higher plant WRKYs34. Although all WRKY TFs bind to the W-box core sequence [TTGAC(T/C)], WRKY33 preferentially binds W-boxes that are within 500 nt of the WRKY33-specific motif [(T/G)TTGAAT])35.The TF WRKY33 regulates the pathogen-inducible biosynthesis of camalexin in Arabidopsis Trp-derived defense metabolism. Specifically, the LINE retrotransposon EPCOT3 has retroduplicated from a WRKY33-TFBS-carrying progenitor and inserted upstream of the newly duplicated gene CYP82C2. Subsequent chromatin remodeling in A. thaliana has led EPCOT3 to become a bona fide enhancer with demonstrated biochemical, regulatory, physiological, and fitness-promoting characteristics by way of WRKY33-binding and pathogen-responsive CYP82C2 transcription, 4OH-ICN biosynthesis, and antibacterial defense.Here we show that a recent, lineage-specific TE exaptation has resulted in the expansion of a core regulon within the framework of A. thaliana, we compared host transcriptional and metabolic responses to the PTI-eliciting bacterial MAMPs flg22, elf26, and fungal MAMP chitosan; the PTI/ETS-eliciting pathogens Pseudomonas syringae pv. tomato DC3000 (Pto DC3000 or Pst); P. syringae pv. maculicola ES4326 (Pma); and the ETI-eliciting pathogens Pst avrRpm1 (Psta), Pst avrRpt2, Pst avrRps4, Pma M2, and Pma avrRpt2 under similar conditions as those of previous studies36. Psm M2 is an ETI-eliciting strain from which the avrRpm1 gene was originally isolated37. Both flg22 and Psta induced genes involved in camalexin, 4OH-ICN, and 4M-I3M biosynthesis, with camalexin and 4OH-ICN biosynthetic genes having a higher level of induction than those of 4M-I3M in Psta-inoculated plants36  have been shown to induce HR-like cell death in Arabidopsis42, a phenomenon commonly observed for ETI16. To our surprise, 300 μg/mL chitosan additionally induced detectable levels of 4OH-ICN  express a wild-type copy of WRKY33 with a C-terminal fusion to 1× flag epitope . We found camalexin and 4OH-ICN levels to be positively correlated among accessions  were used to identify 354 genes that were differentially mutated to high effect in Di-G isogenic accessions Col-0 and Ler-1. wrky33 and Di-G were more susceptible to Pst than their (near-)isogenic relatives and comparable to the 4OH-ICN biosynthetic mutant cyp82C223 -PCR. WRKY33 reportedly binds upstream of 4OH-ICN biosynthetic gene CYP71A12 to a W-box region that also contains three WRKY33-specific motifs49. We additionally observed that Psta-induced WRKY33 bound strongly (greater than fivefold enrichment) upstream of 4OH-ICN biosynthetic genes FOX1 and CYP82C2 to W-box regions that also contain one to three WRKY33-specific motifs  and no support for positive selection acting on CYP82C2/3 enzymes  results in frequent 5′-truncation of retrocopies61. We identified 11 variably truncated retrocopies similar to EPCOT3 throughout the genome  for those with high identity (> 65%) to EPCOT3, and Ta22 or Ta22-LIKE (Ta22L) for the remainder  leaves and A. thaliana cyp82C2 protoplasts transfected with either the A. thaliana or A. lyrata CYP82C2 locus , sharing the W-box and WRKY33-specific motif, whereas EPL2 is less similar (67%) and lacks the WRKY33-specific motif PCR. Compared with EPCOT3  EPL1 then acquired a WRKY33-specific motif by mutation; and (3) EPCOT3 retroduplicated from EPL1 and then acquired epigenetic signatures of an enhancer, thereby allowing selection to act on standing variation rather than de novo mutation for CYP82C2 recruitment into the 4OH-ICN biosynthetic pathway.Of the OT3 Fig.  and lackOT3 Fig. , WRKY33 ely Fig. . Our fin63, and exaptation of TEs into cis-regulatory modules has been hypothesized to be responsible for the rapid transcriptional rewiring in more ancient lineages of vertebrates14. However, few (if any) evolutionarily recent TE exaptation events in vertebrates and higher plants have been demonstrated to have biochemical, regulatory, physiological, and fitness-promoting functions14. With well over a dozen genomes available including the genetic model A. thaliana, the mustard family presents an excellent system for examining such events. In this study, we show that EPCOT3 is a TE-derived enhancer that mediates WRKY33 binding, pathogen-responsive transcription of CYP82C2, synthesis of the species-specific metabolite 4OH-ICN, and pathogen defense . Di-G is the sole member of 1,135 sequenced accessions to have a high-effect single-nucleotide polymorphism (SNP) in WRKY3366, and may have originated from a Ler-0 ethyl methanesulfonate (EMS) mutagenesis screen, whose mutagenesis rate67 is well within the range of ~25,000 SNPs that are not concordant between Di-G and Ler-066  seeds were sown in 12-well microtiter plates sealed with Micropore tape , each well containing ~15 ± 2 seeds and 1 mL of either filter-sterilized 1× Murashige and  Skoog media (pH 5.7–5.8)  or iron-deficient media (amounts per liter): sucrose, 5.0 g; potassium nitrate, 1.9 g; ammonium nitrate, 1.65 g; MES monohydrate, 0.5 g; calcium chloride dihydrate, 0.44 g; magnesium sulfate heptahydrate, 0.37 g; monopotassium phosphate, 0.17 g; myo-inositol, 0.1 g; disodium EDTA, 29.2 mg; manganese sulfate monohydrate, 16.9 mg; zinc sulfate heptahydrate, 8.6 mg; boric acid, 6.2 mg; glycine, 2.0 mg; potassium iodide, 0.83 mg; nicotinic acid, 0.5 mg; pyridoxine hydrochloride, 0.5 mg; sodium molybdate dihydrate, 0.25 mg; thiamine hydrochloride, 0.1 mg; cobalt chloride hexahydrate, 25.0 μg; and copper sulfate pentahydrate, 25.0 μg. On day 9, seedlings were transferred to 6-well microtiter plates, each well containing ~15 seeds and 3 mL Murashige and Skoog or iron-deficient media. For Polyctenium fremontii, surface-sterilized seeds were sown on Murashige and Skoog agar plates. For all other species, surface-sterilized seeds were sown in 6-well plates, each well containing ~15 seeds and 3 mL Murashige and Skoog media. On day 9, media were refreshed prior to bacterial elicitation. Microtiter plates were placed on grid-like shelves over water-filled trays on a Floralight cart  and plants were grown at 21 °C with 60% humidity under a 16 h light cycle (70–80 μE m−2 s−1 light intensity). For ChIP analyses, ~200 surface-sterilized seeds were sown in a 100 × 15 mm petri plate containing 20 mL of 1× Murashige and Skoog media. Media were exchanged for fresh media on day 9. For bacterial infection assays, plants were grown on soil  at 22 °C daytime/18 °C nighttime with 60% humidity under a 12 h light cycle [50 (dawn/dusk) and 100 (midday) μE m−2 s−1 light intensity]. Seed stock information is shown in Supplementary Table For quantitative PCR (qPCR) and high-performance liquid chromatography coupled with diode array detection and fluorescence detection (HPLC-DAD-FLD) analyses, surface-sterilized DEX:WRKY33-flag construct, WRKY33 was PCR-amplified from genomic DNA using the primers WRKY33gXhoF (5′-AACTCGAGAAGAACAAGAACCATCAC-3′) and W33flagSpeR (5′-CGACTAGTCTACTTGTCGTCATCGTCTTTGTAGTCGGGCATAAACGAATCGAAA-3′), and subcloned into the XhoI/SpeI sites of pTA7002 vector68. To generate the DEX:WRKY33-myc construct, WRKY33 was PCR-amplified using the primers WRKY33gXhoF and WRKY33gStuR (5′-AAGGCCTGGCATAAACGAATCGAAAAATG-3′), and subcloned into the XhoI/StuI sites of pTA7002-6x c-Myc vector69. Constructs were introduced into wrky33 and Di-G plants via Agrobacterium tumefaciens-mediated floral dip method70 and transformants were selected on agar media containing 15 μg/mL hygromycin B .To generate the CYP82C2 locus construct, the CYP82C2 upstream and coding sequences were PCR-amplified from A. thaliana genomic DNA using the primers At82C2proXbaF (5′-GCTCTAGAAGCTTCCAATAAAACATTC-3′) and At82C2proBamR (5′-GCGGATCCAGTGGTTTGAGCGTGCAAA-3′), and At82C2geneBamF (5′-GCGGATCCATGGATACTTCCCTCTTTTC-3′) and At82C2geneSmaR (5′-TTCCCGGGCTACTTGTCGTCATCGTCTTTGTAGTCCACATAAAGCCCTTCCTTAAG-3′). Sequences were subcloned into the XbaI/SmaI sites of pBI101 vector71. To generate the AlCYP82C2 locus construct, the AlCYP82C2 upstream and coding sequences were PCR-amplified from A. lyrata genomic DNA using the primers Al82C2proSalF (5′-CGGTCGACTATTCCAGGAGCATACAA-3′) and Al82C2proBglIIR (5′-GGAGATCTAATGTTTTAAAAGTGCAAAAGAG-3′), and Al82C2geneBamHF (5′-GCGGATCCATGGATACATCCCTCTTTTC-3′) and Al82C2geneSmaR (5′-TTCCCGGGCTACTTGTCGTCATCGTCTTTGTAGTCCACAAAAAGTTCTTCCTTAAGAC-3′), and subcloned into the SalI/SmaI sites of pBI101 vector. DEX:WRKY33-flag, CYP82C2, and AlCYP82C2 constructs were introduced into N. benthamiana leaves as previously described23 with the following modifications: leaves were infiltrated with transformed Agrobacterium strains that were grown in lysogeny broth (LB) medium supplemented with 30 μg/mL gentamycin and 50 μg/mL kanamycin to an OD600 of 0.7. Sixteen hours post-Agro-infiltration, leaves were sprayed with 20 μM dex, 0.1% Tween-20, and 1 µM flg22, and incubated for 24 and 30 h. Three 8 mm leaf discs were pooled per sample and snap-frozen for reverse-transcriptase PCR (RT-PCR) analyses. CYP82C2 and AlCYP82C2 constructs were introduced into A. thaliana cyp82C2 via PEG-mediated protoplast transformation72 with the following modifications: 2.5 × 105 protoplasts were transfected with 3 μg of construct for 20 min, recovered in 2.5× volume of W5 solution, elicited with 1 μM flg22 in 1 mL W5 solution for 6 h, and snap-frozen for RT-PCR analyses.To generate the P. syringae pv. maculicola (Pma) M2 , Pma ES4326 (containing no aforementioned effectors), Pma ES4326 avrRpt2, P. syringae pv. tomato DC3000 , Pst avrRpm1, Pst avrRps4, and Pst avrRpt2 from a freshly streaked 3-day-old agar plate was used to inoculate 2 mL of LB medium containing appropriate antibiotics. Strains were grown to log phase, washed in sterile water twice, resuspended in sterile water to OD600 of 0.2, and incubated at room temperature with no agitation for 3 to 6 h, prior to infection. Chitosan  was prepared in 0.1 N acetic acid and neutralized with 0.1 N NaOH to pH 5.8, to a stock concentration of 1.2 mg/mL.A single colony of 600 of 0.013 or treated with 10 μM flg22 , and 150 or 300 μg/mL chitosan. Seedlings were snap-frozen 9 h post infection for ChIP analyses, 12 h post infection for qPCR analyses, and 24–28 h post infection for HPLC-DAD analyses.Hydroponically grown 9-day-old seedlings were inoculated with bacterial strains to ODPst (OD600 = 0.002 or 106 colony-forming units (cfu)/cm2 leaf area) in the presence of 0.01% (v/v) Silwet L-77 for 15 s and incubated on 0.8% (w/v) tissue-culture agar plates at 21 °C under a 16 h light cycle (70–80 μE m−2 s−1 light intensity) for 3 days. Leaves were then surface-sterilized in 70% ethanol for 10 s, rinsed in sterile water, surface-dried on paper towels, and the bacteria were extracted into water, using an 8 mm stainless steel bead and a ball mill (20 Hz for 3 min). Serial dilutions of the extracted bacteria were plated on LB agar plates for cfu counting.Four- to-five-week-old adult leaves were treated with 0.0125% Silwet (Phytotechnology Laboratories) or 0.0125% Silwet and 20 μM dex for 20 s, and incubated on 0.8% (w/v) tissue-culture agar plates on a light cart at 21 °C for 6–8 h. Leaves were then surface-inoculated with EIF4A1 (AT3G13920) and AlEIF4A1 (AL3G26100) housekeeping genes were used to normalize mRNA levels between different samples. Primer sequences and efficiencies are listed in Supplementary Data A. thaliana protoplasts was extracted from snap-frozen samples using 300 µL of Cell Lysis Solution (2% (w/v) SDS, 63 mM sodium citrate, 132 mM citric acid, 1 mM EDTA), 100 µL of Protein-DNA Precipitation Solution , and 300 µL of isopropanol. 2 μg RNA was then reverse-transcribed  and complementary DNA was diluted 7.5-fold. Four microliters of cDNA was used in 20 μL PCR reactions and resulting PCR products were separated on 2% agarose gels. PCR was performed on C1000 thermal cycler (Bio-Rad) with the following thermal cycling program: 95 °C for 3 min; 40 cycles of 95 °C for 10 s, 53 °C for 15 s, and 72 °C for 7 s , 15 s (NbACTIN1), or 21 s (CYP82C2*). Primer sequences are listed in Supplementary Data Total RNA was extracted from snap-frozen seedlings using TRIzol reagent (Invitrogen) and 2.5 µg was reverse-transcribed with 3.75 µM random hexamers (Qiagen), 20 units of M-MuLV (New England Biolabs), and 20 units of ProtoScript II (New England Biolabs) for 1 h at 42 °C and then for 15 min at 70 °C. Resulting cDNA:RNA hybrids were treated with 10 units of DNase I (Roche) for 30 min at 37 °C and purified on PCR clean-up columns (Macherey–Nagel). qPCR was performed with Kapa SYBR Fast qPCR master mix (Kapa Biosystems) and CFX96 real-time PCR machine (Bio-Rad). Biological and technical replicates were performed on the same 384-well PCR plate, and g for 2 min. The supernatant was filtered using a 0.45 μm polypropylene filter plate .Snap-frozen seedlings were lyophilized, weighed, and homogenized using a 5 mm stainless steel bead and ball mill . For phytoalexin analysis, homogenate was extracted with 300 μL 80% (v/v) aqueous methanol containing 0.08% (v/v) formate and 2.5 μL internal standard (200 μM 4-methoxyindole/4M-I [Sigma-Aldrich] in 100% methanol) per mg sample dry weight. Extracts were sonicated for 5 min and centrifuged at 16,000 × cyp79B2 cyp79B3 seedling extract and integrated areas in the UV chromatographs at 260 nm for 4M-I (retention time [RT] = 7.7 min); 340 nm for ICN (RT = 11.5 min); 280 nm for ICN degradation product ICA-ME (RT = 9.5 min), and co-eluting 4OH-ICN degradation products 4OH-ICA and 4OH-ICA-ME (RT = 10.1 min); and 320 nm for camalexin (RT = 12.1 min). For Fig. − (ICN), 174 [M-H]− (ICA-ME), 176 [M-H]− (4OH-ICA), 190 [M-H]− (4OH-ICME), and 201 [M + H]+ , by the peak area at m/z 202 [M-H]− (IBA).Samples were separated on an Ultimate 3000 HPLC  system, using a 3.5 μm, 3 × 150 mm Zorbax SB-Aq column  with the gradient shown in Supplementary Table g for 1 min to remove the water. The homogenate was extracted with 500 μL 70% (v/v) aqueous methanol at 67.5 °C for 10 min and centrifuged at 16,000 × g for 2 min. Added to the supernatant was 3 μL of IS (1.25 mM sinigrin (Sigma-Aldrich) in 80% (v/v) ethanol) per mg sample dry weight. Extract was applied to and incubated on the ion exchanger for 10 min. The sephadex resin was washed three times with 70% (v/v) methanol, three times with distilled deionized water (ddH2O), and two times with 20 mM sodium acetate (pH 5). Twenty microliters of 25 mg/mL aryl sulfatase  was applied to and incubated on the sephadex resin at RT overnight. The plate was centrifuged at 400 × g for 1 min and desulfoglucosinolates were eluted from the sephadex resin by two 100 μL washes with 60% (v/v) methanol and two 100 μL washes with ddH2O. Eluate volume was reduced to 250–350 μL using an evaporator.For glucosinolate extraction, a 96-well 0.45 μm polyvinylidene fluoride (PVDF) filter plate  was charged with 45 mg DEAE Sephadex A25 (GE Heathcare) and 300 μL of water per well, and equilibrated at room temp for 2 h. Prior to sample homogenization, the plate was centrifuged at 400 × 73.Samples were separated on an Ultimate 3000 HPLC system, using a 3.5 μm, 3 × 150 mm Zorbax SB-Aq column with the gradient shown in Supplementary Table wrky33/DEX:WRKY33-flag nuclear extracts, approximately two hundred and ten 9-day-old seedlings were inoculated with Psta to OD600 of 0.013 and co-treated with mock solution of dimethyl sulfoxide (M) or 20 μM dex (D) for 9 h. Total protein was extracted in 25 mL of Extraction buffer 1  formaldehyde solution and a 5 min quenching step with 2 M glycine, seedlings were washed three times with deionized water, vacuum-dried, and snap-frozen with liquid nitrogen. Following frozen homogenization, the homogenate was filtered once through a 70 µm mesh  and a 0.45 µm filter (EMD Millipore). Filtered homogenate was then washed once in 500 µL of Extraction buffer 2  and resuspended in 300 µL of Extraction buffer 3  prior to sucrose centrifugation. Following nuclear extraction, samples were resuspended in 125 µL of Nuclei Lysis buffer , and 250 µL of ChIP dilution buffer , sonicated in a Covaris S2 sonicator  using 10% duty, 7% intensity, 200 cycles per burst for a total time of 11 min, and centrifuged at 16,000 × g for 10 min at 4 °C to precipitate SDS. ChIP was performed using Anti-FLAG M2 Affinity Gel (Sigma-Aldrich). Beads were pre-treated with 0.1% (w/v) non-fat milk in 1× phosphate-buffered saline (PBS) and 0.5 mg/mL sheared salmon sperm DNA (Invitrogen). Following de-crosslinking, DNA samples were phenol-chloroform-extracted, diluted to the same OD260 concentration, and 1.5 μL was used in a 15 μL PCR reaction.For ChIP experiments on EPL2, qPCR analysis was additionally performed to confirm absence of amplicons in ChIP samples. RLU counts at the 25th cycle were used for quantification. Primer sequences are listed in Supplementary Data PCR analysis was performed on nuclear extracts prior to antibody incubation (input) and after ChIP. PCR conditions were as follows: 95 °C for 3 min; 40 cycles of 95 °C for 15 s, 53 °C for 15 s, and 72 °C for 1 min; 72 °C for 5 min. Densitometric determination of signal intensity in each ChIP and input sample was calculated using ImageJ. Fold enrichment was determined by calculating the ratio of PCR product intensities in ChIP D/M to Input D/M. In cases where amplicons were absent, an arbitrary value of 10 was assigned. For g for 8 min to precipitate insoluble material.Total protein was extracted from snap-frozen seedlings into 80 µL of extraction buffer  using a 5 mm stainless steel bead and ball mill (20 Hz for 3 min). Samples were centrifuged briefly, incubated at 95 °C for 10 min, and centrifuged at 12,000 × Five (for WRKY33-flag) or 15 µL (for WRKY33-myc) of extract was loaded onto a 10% SDS-PAGE gel and the separated proteins were transferred to PVDF membrane , stained with Ponceau S for labeling of total protein, and probed with either FLAG M2  or c-Myc 9E10  antibodies diluted 1:1000 or 1:750, respectively, in 1× PBS containing 5% (w/v) non-fat milk.75. To generate phylogenetic maximum likelihood (ML) trees, sequences were aligned using MUSCLE in MEGA776 and the JTT model  or Tamura-Nei model . Sequences for all genes with the description “non-LTR retrotransposon family (LINE)” (n = 263) were batch-downloaded from TAIR (https://arabidopsis.org). Of these, sequences containing intact reverse-transcriptase domains  were used for subsequent phylogenetic analysis  was performed for each Branch site model to be tested (model = 2 and NSsites = 2), where omega was fixed to 1. Branch site models were then tested with unfixed omega. Likelihood ratio tests were performed by comparing critical values and degrees of freedom between each unfixed Branch site test and either the M1a test or the corresponding fixed-omega test. Pre-defined branches with P-values < 0.05 for both tests were regarded as under positive selection . Promoter alignment plots were generated using mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml)78.Epigenetics data were obtained from published workFurther information on research design is available in the Supplementary InformationPeer ReviewReporting SummaryDescription of Additional Supplementary FilesSupplementary Dataset 1Supplementary Dataset 2Source Data


Fusaruside is an immunomodulatory fungal sphingolipid which has medical potentials for treating colitis and liver injury, but its poor natural abundance limits its further study.Pichia pastoris that was based on polycistronic expression. Two fusaruside biosynthesis genes (Δ3(E)-sd and Δ10(E)-sd), were introduced into P. pastoris to obtain fusaruside producing strain FUS2. To further enhance the yield of fusaruside, three relevant biosynthetic genes (Δ3(E)-sd, Δ10(E)-sd and gcs) were subsequently introduced into P. pastoris to obtain FUS3. All of the biosynthetic genes were successfully co-expressed in FUS2 and FUS3. Compared to that produced by FUS2, fusaruside achieved from FUS3 were slightly increased. In addition, the culture conditions including pH, temperature and methanol concentration were optimized to improve the fusaruside production level.In this study, we described a synthetic biology approach for fusaruside production by engineered P. pastoris fusaruside production system was developed by introducing the biosynthetic genes linked by 2A peptide gene sequences into a polycistronic expression construct, laying a foundation for further development and application of fusaruside.Here a novel The online version of this article (10.1186/s12896-019-0532-8) contains supplementary material, which is available to authorized users. Fusarium endophytes [Fusarium sp., on the other hand fusaruside are trace quantity in natrual fungi. Only 24 mg fusaruside could be obtained from about 200 g crude ethyl acetate extract [Fusarium into metabolic engineered Pichia pastoris, to produce a large number of clinically useful fusaruside.Fusaruside is a kind of sphingolipid, isolated as a minor compound from dophytes , 2. It hdophytes –5. Howev extract . Additio extract . As an aP. pastoris harbours cerebroside D [F. graminearum is the missing of C3-double bond on the N-Acyl chain. Cerebroside D can be metabolized by 2-hydroxy fatty N-acyl-delta3(E)-desaturase (Δ3(E)-SD) to form cerebroside B [P. pastoris, it is highly plausible to target fusaruside biosynthesis through metabolic engineering of the yeast by co-overexpressing Δ10(E)-SD and Δ3(E)-SD -SD and Δ3(E)-SD in yeast expression host systems to obtain fusaruside producing strain, then we co-expressed GCS with Δ10(E)-SD and Δ3(E)-SD to enhance the yield of fusaruside. The strategy adopted was to engineer yeast using a self-cleaving 2A peptide based vector system to realize synchronous production of the enzymes.Sac I, SnaB I and Not I were purchased from Takara Bio Inc., Japan, and used as detailed by the manufacturer. Yeast nitrogen base w/o amino acids (YNB), D-sorbitol, Kanamycin monosulfate, Zeocin and geneticin sulfate (G-418S) were obtained from Solarbio, Beijing, China. Oxoid™ peptone and yeast extract were purchased from Thermo Scientific, Germany.Restriction enzymes P. pastoris was grown in yeast peptone dextrose medium  or buffered complex glycerol medium . P. pastoris was induced in buffered complex methanol medium . YPD plates containing 1 M Sorbitol and 2.5 mg/mL geneticin sulfate were used for selection of positive strains containing the pPIC3.5 K expression vector. E. coli was cultivated in LB medium . F. graminearum was also grown in YPD medium.F. graminearum PH-1 (NRRL 31084) was cultured on YPD medium for 72 h at 28 °C with shaking of 150 rpm. About 3 to 5 g wet fungal mycelium was collected by filtration on sterile filter paper and used for RNA extraction with the RNAeasy Mini Kit (Qiagen) following the manufacturer’s recommendations.For RNA isolation, P. pastoris GS115 was grown on YPD medium for 24 h at 30 °C and 220 rpm. The yeast cells were collected by centrifugation with 5000 rpm and 5 to 10 g (wet weight) was used for RNA extraction using the RNAeasy Mini Kit.F. graminearum Δ3(E)-sd gene (GenBank accession: XM_383758.1) was amplified from cDNA by PCR using primers delta3-F (5′-TACGTAGCCACCATGGCCGAACACCTCGTCTTC) and delta3-R (5′-CTGCCTCTTAAACTTCTTC) which contain restriction sites  for SnaB I.The cDNA was obtained by RT-PCR from total RNA, using PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa). The DNA fragment encoding the F. graminearum Δ10(E)-sd gene (GenBank accession: XP_390021.1) was amplified by PCR using primers delta10-F (5′-ATGGCGCATAGCTCTTTCGTT) and delta10-R1  to co-express Δ3(E)-SD and Δ10(E)-SD, or amplified using primers delta10-F and delta10-R2 (5′-GTGATGAGAGAGATCACC) to co-express three enzymes, Δ3(E)-SD, Δ10(E)-SD and GCS. The P. pastoris gcs gene (GenBank accession: AF091397) was amplified using primers gcs-F (5′-ATGTCACAACTCAGACCCAG) and gcs-R . The 2A polypeptide from the aphthovirus foot-and-month disease virus (FMDV) was selected and synthesized as part of a 66 bp synthetic sequence (5′-GGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCT) and cloned into pUC19 by BioSune Inc. (Shanghai).The 2+, dNTP and polymerase), 0.25 μM each primers and 10 ng template in a final volume of 50 μL.PCR was performed with PrimeSTAR Max DNA Polymerase (TaKaRa) under the following cycling parameters: 30 s at 98 °C for a first denaturation step, 30 cycles of 10 s at 98 °C, 15 s at 55 °C and 30 s at 72 °C, and 1 cycle of a final extension step at 72 °C for 5 min. Reaction mixtures contained 1× PrimeSTAR Max Premix buffer -sd, Δ10(E)-sd and 2a were firstly amplified with delta3-F/delta3-R, delta10-F/delta10-R1 and 2a-F1 (5′- GAAGAAGTTTAAGAGGCAGGGATCCGGAGCCACGAAC)/2a-R1(CGAAAGAGCTATGCGCCATAGGACCGGGGTTTTCTTC) primers, respectively. The products were then fused by splicing overlap extension PCR (SOE-PCR) using delta3-F/delta10-R1 as primer. Finally, the fused gene Δ3(E)-sd–2a–Δ10(E)-sd were sub-cloned into pPIC3.5 K, using SnaB I and Not I sites -sd–2a–Δ10(E)-sd, gcs and 2a were amplified with delta3-F/delta10-R2, gcs-F/gcs-R and 2a-F2 (5′-GGTGATCTCTCTCATCACGGATCCGGAGCCACGAAC)/2a-R2(5′-GGGTCTGAGTTGTGACATAGGACCGGGGTTTTCTTC) primers, respectively. Subsequently, the products were fused by SOE-PCR using delta3-F/gcs-R as primer. The fused products Δ3(E)-sd–2a–Δ10(E)-sd–2a–gcs were finally sub-cloned into pPIC3.5 K, using SnaB I and Not I sites . The electroporational parameters were set as 200 Ω, 25 μF and 1.5 kV. After pulse, 500 μL of ice-cold 1 M sorbitol was added immediately and incubated at 28 °C for 1 h. After that, another 500 μL of YPD was added for further 2 h of regeneration at the same temperature. After 5~7 days, positive transformants were selected on YPD plates containing 2.5 mg/mL Geneticin 418, and then lysed using a combination of enzyme, freezing and heating according to a simple protocol described in the literature [P. pastoris to be used as negative control.To obtain stable expression strains, the genes in expression vectors  were usually transformed to e genome . The proe genome . Firstlyterature . The celhttp://web.expasy.org/compute_pi/.Positive clones were cultured in 10 mL BMGY medium in a 100 mL flask at 30 °C with shaking of 200 rpm until OD600 reaching 12~16. At room temperature, the cultures were collected by centrifugation at 5000 rpm for 5 min. The cell pellets were suspended in 20 mL BMMY medium and grew under the same conditions . Methanol was added to the culture every 24 h to a final concentration of 0.5% (v/v) to induce proteins co-expression. At the same time, 1 mL of culture medium was collected every 24 h after initiating induction. The yeast cells were immediately frozen and stored for further sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) analysis. The theoretical molecular weights (MW) of the biosynthetic enzymes were calculated using the website P. pastoris cells were treated as described [P. pastoris cells were firstly suspended in 5 mL of H2O and boiled in a water bath for 15 min. Then, the cells were sedimented by centrifugation at 5000 rpm for 5 min and used to extract sphingolipids. After that, 10 mL of dichloromethane/methanol (v/v = 1:1) was added and shaken overnight at 4 °C to extract the yeast sphingolipids. The next day, another 10 mL of dichloromethane/methanol (v/v = 2:1) was added and shaken for at least 4 h at 4 °C. Subsequently, the 20 mL of sphingolipid extract was washed three times with dichloromethane/methanol/0.45% NaCl (v/v = 8:4:3). The organic phase  was collected and the solvents were removed by a rotary evaporator.After 84 h of induction, escribed . ApproxiSphingolipids extracts in organic solvents were analyzed and quantified by liquid chromatography technique coupled with mass spectrometry (LC-MS) as our previous study . To idenP. pastoris including methanol concentration, pH and temperature were optimized. All experiments were performed in triplicates.In order to increase the production of fusaruside in yeast, culture and induction conditions of engineered Δ3(E)-sd and Δ10(E)-sd genes, or consisting of Δ3(E)-sd, Δ10(E)-sd and gcs genes, separated by the 2A sequences were cloned into the P. pastoris expression vector pPIC3.5 K, which is under the regulation of the AOX1 promoter -sd − Δ10(E)-sd and pPIC3.5 K − Δ3(E)-sd − Δ10(E)-sd − gcs were linearized with Sac I and transformed into P. pastoris GS115 competent cells by electroporation, respectively. Positive transformants were selected by the ability to grow on YPD plates containing 2.5 mg/mL of Geneticin 418, due to the presence of Kan gene in the pPIC3.5 K vector. The genomic DNA of P. pastoris recombinants was isolated to perform PCR verification using delta3-F/delta10-R1 or delta3-F/gcs-R as primers. One band corresponding to the size of the Δ3(E)-sd gene plus the Δ10(E)-sd gene (~ 3000 bp) were obtained in the chromosome of P. pastoris intergrated by pPIC3.5 K − Δ3(E)-sd − Δ10(E)-sd -sd − Δ10(E)-sd − gcs, a band corresponding to the size of three genes (~ 6000 bp) were also detected  cerebroside B and 0.39 ± 0.04 mg/g fusaruside. The cerebroside B and fusaruside produced by strain FUS3 were 1.25 ± 0.22 and 0.52 ± 0.06 mg/g, respectively. The identity of cerebroside B and fusaruside isolated from P. pastoris transformants were also confirmed by NMR spectroscopy. The 1H NMR spectra of cerebroside B .The strain of FUS3, co-expressing three enzymes, was investigated in BMMY media at different pH levels . As shown in Fig. Fusarium sp. endophytes has become the main bottleneck restricting its further pharmacological research. In this work we sought to produce fusaruside in engineered P. pastoris by co-expressing biosynthetic genes, Δ3(E)-SD, Δ10(E)-SD and GCS. A self-processing 2A peptide from FMDV was applied to construct polycistronic systems for gene co-expression in yeast, due to its capacity of simultaneous and efficient expression of multiple genes [Aspergillus nidulans strains using 2A peptide-based system [P. pastoris [Fusaruside is a potential pharmaceutical molecule due to its ability to treat liver injury and colitis through selective immunosuppressive effect. But its poor natural abundance in le genes –19. 2A sle genes . Similard system . Geier epastoris .P. pastoris has been previously described [P. pastoris [Although the individual expression of Δ3(E)-SD and Δ10(E)-SD in escribed , 10. We pastoris . As we eP. pastoris expression systerm [AOX1 promoter [P. pastoris cell growth is 30 °C. But in our previous study, the content of fusaruside may increase at lower temperature to resistant cold environment [Pichia cells declined too much at 15 °C, and therefore fusaruside production was affected. Finally, at optimum condition  a high level (0.74 ± 0.14 mg/g) of fusaruside in yeast FUS3 was achieved after 120 h induction. Compared to the yield of original Fusarium sp. (0.12 mg/g after 10 d) [P. pastoris was satisfactory.It has been demonstrated that growth and induction conditions are critical parameters for  systerm –25, thuspromoter , methanopromoter –28. Hereironment . We askeThosea asigna virus [Saccharomyces cerevisiae [Δ3(E)-sd, Δ10(E)-sd and gcs genes.To further improve the yield of fusaruside, there are still several measures can be done. Except the 2A sequence from FMDV used in this work, there are some other 2A sequences, such asthe P2A sequence from porcine teschovirus-1 and T2A from na virus . It has revisiae . Thus direvisiae . This giΔ3(E)-sd, Δ10(E)-sd and gcs, via 2A peptide sequences. Thus, the engineered P. pastoris yeast can generate fusaruside via glycosphingolipid pathway, and this opens for the use of yeast as a cell factory for production of cerebrosides in future.In summary, we reconstituted a heterologous fusaruside biosynthetic pathway by linking three genes, Additional file 1:Figure S1. Identification of co-expression plasmid by digestion. a. M: DNA marker; 1: product of double enzyme digestion of pPIC3.5 K − Δ3(E)-sd − Δ10(E)-sd. b. M: DNA marker; 1: product of double enzyme digestion of pPIC3.5 K − Δ3(E)-sd − Δ10(E)-sd − gcs. Figure S2. Identification of transformants by PCR. a. M: DNA marker; 1–2: PCR products with primer delta3-F/ delta10-R. b. M: DNA marker; 1–2: PCR products with primer delta3-F/gcs-R. Figure S3.1H NMR of cerebroside B in CDCl3 (400 MHz). Figure S4.1H NMR of fusaruside in CDCl3 (400 MHz). (DOCX 341 kb)


Additionally, the structure of 1 was ambiguously verified by single-crystal X-ray diffraction, while the structure of 2, which features a novel 8,16-epoxy motif, was validated by quantum chemical calculation of NMR parameters and ECD spectrum. Moreover, 1 and 4 were found to exhibited anticoagulant activity, while 3 and 4 were found to possess anti-platelet aggregation activity.Four new rearranged 6/6/5/6-fused lanostane-type triterpenoids, kadcoccitanes A–D (The online version of this article (10.1007/s13659-019-0203-4) contains supplementary material, which is available to authorized users. Schisandra and Kadsura, are invaluable sources of triterpenoids +, calcd 509.3237), demonstrating eight degrees of unsaturation. The 1H-NMR data of 1 , one doublet methyl (δH 1.16) and two olefinic protons (δH 5.29 and 6.07). Its 13C NMR and DEPT spectra revealed 30 carbon resonances  implied the carbonylation of C-3; The HMBC correlations from H-12 and H2-16 to C-13 (δC 77.0), in combination with the HMBC correlations from H2-18 (δH 4.16) to C-12, C-13 and C-17 ascertained the substitution of a hydroxyl group at C-13 and C-18, respectively. The HMBC correlations from H3-27 to C-24 (δC 142.9), C-25 (δC 129.0) and C-26 (δC 170.9), and from H-24 (δH 6.07) to C-26 implied the existence of the C-24/C-25 double bond and carboxyl group at C-26. The COSY correlation of H-11 (δH 5.29)/H-12 and the HMBC correlations from H-12 and H3-19 to C-9 (δC 154.4) suggested the presence of Δ9,11 double bond. By analyzing its ROESY spectrum, the cross-peak of H-24/H3-27 indicated that the Δ24,25 double bond took Z-geometry. However, the configuration of C-13 couldn’t be determined due to the lack of HO-13 signal in the 1H NMR spectrum. Fortunately, the crystals of 1 were obtained and subjected to the X-ray diffraction analysis through CuKα radiation, which determined its absolute configuration to be 5R,8S,10S,12R,13R,14S,17R, and 20R (Flack parameter = 0.06(2)) (CCDC:1903695)  was obtained as white amorphous powder. The molecular formula C30H42O6 was determined by HRESIMS , corresponding to ten indices of hydrogen deficiency. The 1H-NMR spectrum showed proton signals which could be ascribed to six singlet methyls , one doublet methyl (δH 0.92) and two olefinic protons (δH 5.94 and 6.15)  as that of compound 1, as well as a similar A/B ring system except that C-8 (δC 92.8) was oxygenated, as revealed by the 1H-1H COSY correlations of H-5/H2-6/H2-7, together with the HMBC correlations from H2-6 and H2-7 to C-8. Additionally, the HMBC correlations from olefinic H-11 (δH 5.94) to C-12 (δC 88.2), from H3-28 to C-15 (δC 78.1), and from H-15 (δH 4.33) to C-16 (δC 80.0) demonstrated that C-12, C-15, and C-16 were all oxygenated. Besides, the correlations from H3-18 and H-16 (δH 4.70) to C-13 (δC 137.7) and C-17 (δC 136.8) suggested the presence of the C-13/C-17 double bond. Most importantly, the correlation of H-16/C-8, the intensity of which was nearly as strong as that of the H-15/C-8 correlation, was observed in the HMBC spectrum. Hence, in consideration of the 4-bond distance between H-16 and C-8 if following the H-16/C-16/C-15/C-14/C-8 path, an epoxy was tentatively constructed between C-8 and C-16.Careful analysis of the HMBC and tra Fig.  of 2 dem2, the H-15/H3-28α correlation in the ROESY spectrum of 2 -2 (2a) and -2 (2b)  were subjected to quantum calculations of NMR chemical shifts at MPW1PW91-SCRF/6-31 + G//M06-2X-D3/def2-SVP level of theory in chloroform with IEFPCM solvent model (Tables S2 and S3). As a result, the calculated NMR shifts of 2b were found to be in better agreement with their experimental counterparts, as indicated by parameters including R2, MAE, CMAE, as well as the DP4 + probability (Table 2b was run at B97-2/pcJ-1 level with IEFPCM solvent model in chloroform (Table S5), the theoretical SSCC of 3JH-15/H-16 (0.5 Hz) could clearly explain the aforementioned splitting pattern of H-15 and H-16 in the 1H NMR spectrum, while the predicted SSCCs of 3JH-16/C-8 (7.0) could well account for the strong H-16/C-8 correlation in the HMBC spectrum. Thus, the planar structure as well as the relative configuration of 2 was established as depicted in Fig. 2 as 5R, 8S, 10S, 12R, 14R, 15S, 16R and 20S  represented the first example of 8,16-epoxy-14(13 → 12)-abeolanostane triterpenoid featuring an unusual 20S configuration.As for the stereochemistry of 3), white amorphous powder, possessed a molecular formula of C30H46O6 supported by the HRESIMS , with eight indices of hydrogen deficiency. Five singlet methyls , one doublet methyl (δH 1.11) and one olefinic proton (δH 6.12) were observed from its 1H NMR spectrum , from H-11 (δH 5.14) to C-10 and C-14, and from H-8 to C-11 (δC 70.1) , and from H-11 and H2-18 to C-13 (δC 137.7)  was isolated as white amorphous powder. Its HRESIMS data  indicated that it possessed a molecular formula of C30H48O4, suggesting seven degrees of unsaturation. The 1H NMR spectrum showed resonances for six singlet methyls , one doublet methyl (δH 1.19) and two olefinic protons (δH 5.56 and 6.13)   and C-17 manifested that the hydroxymethyl of C-18 was replaced by a methyl  Fig. . MoreoveC-13 δC 7.7 and C-C-13 δC 7.7 and C-K. coccinea indicated that the secondary metabolites from the roots generally possess higher degrees of oxidation than those obtained from its stems, which may be mainly attributed to the abundant oxidases existing in the roots. However, to arrive at more definite conclusion, more in-depth research still needs to be undertaken. In addition, compounds 1, 3 and 4 were screened for the bioactivity against platelet aggregation induced by colloid. The result suggested that the inhibition ratios of 3 and 4 were 12.4 ± 12.5% and 19.4 ± 14.4% (p < 0.05), respectively, under the concentration of 100 µM. Compounds 1 and 4 were tested for anticoagulant activity. The result showed that their IC50 values were 37.8 and 31.5 µM, respectively.The phytochemical investigations on δ) are expressed in ppm. HRESIMS was performed on an API QSTAR Pulsar i spectrometer. Melting point was recorded on an RDY-1B micro melting point apparatus. UV spectra were obtained on a Shimadzu UV-2401PC spectrophotometer. Optical rotations were measured in MeOH with a JASCO P-1020 polarimeter or an Autopol VI, Serial #91058. IR spectra were obtained on a Bruker Tensor-27 FT-IR spectrometer using KBr pellets. Column chromatography (CC) was performed with silica gel . Analytical HPLC was performed on an Agilent 1260 liquid chromatograph with a Zorbax SB-C18 (4.6 mm × 250 mm) column. Semi-preparative HPLC was performed on an Agilent 1200 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 250 mm) column or a COSMOSIL Cholester (10ID × 250 mm) column. Fractions were monitored by thin layer chromatography, spots were visualized by UV light (254 nm and 365 nm) and by heating silica gel plates sprayed with 10% H2SO4 in EtOH. All solvents used in column chromatography were distilled.1D and 2D NMR spectra were recorded on Bruker AV III 500 MHz spectrometer using TMS as the internal standard. Chemical shifts (K. coccinea (Lem.) A. C. Smith were collected from Jingzhou Miao and Dong Autonomous County in Hunan Province, People’s Republic of China, in June 2016 and identified by Prof. Heng Li at Kunming Institute of Botany. A voucher specimen (KIB 2016062101) has been deposited in State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.The roots of K. coccinea were soaked with industrial alcohol (25 L) for four times, 3 days each time at room temperature. The total extracting solution was sequentially condensed to a certain extent under the condition of heating (40 °C) and reduced pressure in vacuo. Then the concentrate was suspended in water . The remaining alcohol was continuously evaporated under the same condition. After that, the sample was extracted with EtOAc and n-BuOH  for four times, respectively.The air-dried and powdered roots (15.0 Kg) of 3/Me2CO gradiently (1:0 to 0:1) to obtain seven fractions A–G Fraction D (68 g) was chromatographed by RP-C18 using a gradient mobile phase of MeOH/H2O (30:70 to 90:0) to obtain fractions D1–D7.The EtOAc extract (1.8 Kg) was subjected to a silica gel column chromatography eluted with CHCl2CO (1:0–0:1) to afford fractions D51–D55. Fr. D53 (1 g) was subjected to Sephadex LH-20 chromatography using MeOH as mobile phase to give fractions D531–D535. Crystals  were separated from the solution of Fr. D532 (180 mg). Fr. D533 (230 mg) underwent preparative HPLC  and multiple semi-preparative HPLC  to yield compound 2 (9.5 mg).Fr. D5 (8 g) was chromatographed by silica gel with a gradient elution of PE/Me2CO (1:0–0:1) to afford fractions D61–D65. Fr. D64 (1.5 g) was subjected to Sephadex LH-20 chromatography eluted by MeOH and then purified by repeated semi-preparative HPLC  to yield compound 3 (3.3 mg). Fr. D65 (980 mg) was chromatographed by Sephadex LH-20 eluted with MeOH to give fractions D651–D655. Fr. 653 (62 mg) was further purified by repeated semi-preparative HPLC  to yield compound 4 (7.4 mg).Fraction D6 (10 g) was chromatographed by silica gel with a gradient elution of PE/Me1): Colorless acicular crystals, melting point: 213 °C. λmax (log ε) 206 (3.9) nm. IR (KBr) νmax 3440, 2936, 1703, 1638, 1421, 1241, 1035, 886, 647 cm−1. CD (MeOH): 205 nm (Δε = − 0.75), 215 nm (Δε = − 1.03). Positive ESIMS m/z 509 [M + Na]+. 1H and 13C NMR data in pyridine-d5, see Tables 1H and 13C NMR data in CDCl3, see Table S1.Kadcoccitane A : ColorleCrystal data for 1 C30H46O5, M = 486.67, a = 10.6452(2) Å, b = 27.2411(5) Å, c = 28.4264(5) Å, α = 90°, β = 90°, γ = 90°, V = 8243.3(3) Å3, T = 100(2) K, space group P212121, Z = 12, μ(CuKα) = 0.618 mm−1, 49684 reflections measured, 14281 independent reflections (Rint = 0.0301). The final R1 values were 0.0336 (I > 2σ(I)). The final wR(F2) values were 0.0874 (I > 2σ(I)). The final R1 values were 0.0338 . The final wR(F2) values were 0.0876 . The goodness of fit on F2 was 1.087. Flack parameter = 0.06(2).2): White amorphous powder. λmax (log ε) 216 (4.1) nm, 196 (4.1) nm, 206 (4.0) nm. IR (KBr) νmax 3445, 2967, 2934, 1693, 1640, 1458, 1383, 1048, 581 cm−1. CD (MeOH): 222 nm (Δε = 2.14), 244 nm (Δε = − 0.38). Positive ESIMS m/z 521 [M + Na]+. 1H and 13C NMR data in pyridine-d5, see Tables Kadcoccitane B mentclass2pt{minimpt{minim23): White amorphous powder. λmax (log ε) 207 (4.0) nm. IR (KBr) νmax 3432, 2929, 1701, 1638, 1457, 1383, 1264, 1043, 893, 582 cm−1. CD (MeOH): 196 nm (Δε = − 1.48), 207 nm (Δε = + 0.25), 225 nm (Δε = − 0.41). Negative ESIMS m/z 501 [M–H]−. 1H and 13C NMR data in pyridine-d5, see Tables Kadcoccitane C mentclass2pt{minimpt{minim34): White amorphous powder. λmax (log ε) 208 (4.0) nm. IR (KBr) νmax 3433, 2936, 1695, 1638, 1458, 1337, 1252, 1037, 859, 580 cm−1. CD (MeOH): 208 nm (Δε = 0.15), 222 nm (Δε = − 0.25). Positive ESIMS m/z 495 [M + Na]+. 1H and 13C NMR data in pyridine-d5, see Tables Kadcoccitane D mentclass2pt{minimpt{minim42a and 2b was initially performed in Spartan’16  using the Monte Carlo algorithm and Merck molecular force field (MMFF). To avoid losing relevant conformations during the conformational search stage, the “set torsions” function was used to give all rotatable bonds a six fold sampling, as well as to allow the atoms on the aliphatic ring to flip. Maximum 20000 conformers were examined for each diastereoisomer, and those obtained conformers within 20 kcal/mol were kept (1000 ones for each isomer)._ENREF_1Conformational analysis of These conformers were subjected to semiempirical geometry optimization using the GFN2-xTB method  implemenPi is the population of the ith conformer; ni the number of molecules in ith conformer; ΔG is the relative Gibbs free energy ; T is the temperature, usually the room temperature (298.15 K); R is the ideal gas constant (0.0019858995); Q is the partition function. Those conformers accounting for over 98% population were subjected to subsequent calculations.The above screened conformers were subjected to DFT geometry optimization at M06-2X-D3/def2-SVP level of theory. Frequency analyses of all optimized conformers were undertaken at the same level of theory to ensure they were true local minima on the potential energy surface. Then, energies of all optimized conformers were evaluated at M06-2X-D3/def2-TZVP level of theory. Gibbs free energies of each conformers were calculated by adding “Thermal correction to Gibbs Free Energy” obtained by frequency analysis to electronic energies obtained at M06-2X-D3/def2-TZVP level of theory. Room-temperature (298.15 K) equilibrium populations were calculated according to Boltzmann distribution law:δcal = σTMS − σcal), where the σTMS was the shielding constant of TMS calculated at the same level. For each possible candidate, the parameters a and b of the linear regression δcal = aδexp + b; the correlation coefficient, R2; the mean absolute error (MAE) defined as Σn |δcal − δexp|/n; the corrected mean absolute error, CMAE, defined as Σn |δcorr − δexp|/n, where δcorr = /a, were calculated [NMR shielding constants were calculated with the GIAO method at mPW1PW91-SCRF/6-31 + G level with IEFPCM solvent model in chloroform solvent. The shielding constants obtained were converted into chemical shifts by referencing to TMS at 0 ppm . The supernatant was thus collected as platelet rich plasma (PRP). The residual blood was sequentially centrifuged  and supernatant was collected as platelet poor plasma (PPP). The platelet count of PRP was adjusted to 500 × 109 L−1 based on PPP [The experimental blood was extracted from the ears of Japanese white rabbits and conserved in vacuum blood-collection tubes with sodium citrate (whole blood: sodium citrate = 9:1). The blood-collection tubes were turned upside down gently to ensure homogeneous mixing of blood and anticoagulant and then centrifuged  was added into the cuvettes. The curve of platelet aggregation was recorded and the maximum aggregation rate were accordingly calculated. The inhibition rate of the test samples against rabbit platelet aggregation induced by colloid could also be calculated. The formula is as follows: R = (A − B) * 100%/A. R: Inhibition rate, A: The maximum aggregation of solvent, B: The maximum aggregation of test sample (or reference substance).Two cuvettes, one with stir bar and the other without stir bar, were put in the heater of the platelet aggregation apparatus, heated at 37 °C for 10 min. Then 250 The test compounds were diluted with DMSO to 10 mM and then diluted with 0.02 M Tris-HCl (pH 7.4) with 5% Tween 80 to 1 Mm. The positive control was Low Molecular Weight Heparin (LMWH) and the blank control was 0.02 M Tris-HCl (pH 7.4) with 5% Tween 80 and 10% DMSO. The solution of the test sample or reference substance was added into the cuvette preheated at 37 °C and then the control plasma was also added into it. After heating at 37 °C for 2 min, the TT (thrombin time) reagent preheated at 37 °C was also added into it. The clotting time was recorded afterwards.1–4, computational data of 2 are available. Supplementary material 1 (DOCX 13247 kb)Supplementary data associated with this article including 1D and 2D NMR, ESIMS, HRESIMS, UV, IR, CD and OR of Below is the link to the electronic supplementary material.


The unsolvated 2-hydroxybenzoate, pyridine-3-carboxylate and 2-hydroxy-3,5-dinitrobenzoate salts, C11H17N2O+·C7H5O3− (V), C11H17N2O+·C6H4NO2− (VI) and C11H17N2O+·C7H3N2O7− (VII), respectively, are all fully ordered: the components of (V) are linked by multiple N—H⋯O hydrogen bonds to form a chain of rings; those of (VI) are linked into a three-dimensional framework by a combination of N—H⋯O, C—H⋯O and C—H⋯N hydrogen bonds and those of (VII), where the anion has a structure reminiscent of the picrate anion, are linked into a three-dimensional array by N—H⋯O and C—H⋯O hydrogen bonds. The hydrogensuccinate and hydrogenfumarate salts, C11H17N2O+·C4H5O4− (VIII) and C11H17N2O+·C4H3O3− (IX), respectively, are isomorphous, and both exhibit disorder in the anionic component: N—H⋯O and O—H⋯O hydrogen bonds link the ions into sheets, which are further linked by C—H⋯π(arene) interactions. The anion of the hydrogenmaleate salt, C11H17N2O+·C4H3O3− (X), contains a very short and nearly symmetrical O⋯H⋯O hydrogen bond, and N—H⋯O hydrogen bonds link the anions into chains of rings. The ions in the trichloroacetate salt, C11H17N2O+·C2Cl3O2− (XI), are linked into simple chains by N—H⋯O hydrogen bonds. In the hydrated chloranilate salt, 2C11H17N2O+·C6Cl2O42−·2H2O (XII), which crystallizes as a non-merohedral twin, the anion lies across a centre of inversion in space group P21/n, and a combination of N—H⋯O and O—H⋯O hydrogen bonds generates complex sheets. Comparisons are made with the structures of some related compounds.Twelve 4-(4-methoxyphenyl)piperazin-1-ium salts containing organic anions have been prepared and structurally characterized. The monohydrated benzoate, 4-fluorobenzoate, 4-chlorobenzoate and 4-bromobenzoate salts, C N-(4-methoxyphenyl)piperazine (MeOPP) has emerged as a new addition to the range of designer recreational drugs, and considerable effort has been invested in the development of methods for the detection both of MeOPP itself and of its metabolites N-(4-hydroxyphenyl)piperazine and 4-hydroxyaniline piperazine derivatives, and we have recently reported the synthesis and structures of a series of 1-aroyl-4-(4-methoxyphenyl)piperazines –(XI) are all 1:1 salts, but in (XII)et al., 1987et al., 1987et al., 2006et al., 2017et al., 2017abIn the anion of compound (VII)et al., 1987A)–(D) ) Fig. 13 are requet al., 1990et al., 1995n, n + n represents an integer in each case –(IV), the ions are linked by a combination of N—H⋯O and O—H⋯O hydrogen bonds Table 1 to form e Fig. 14. In each2 hydrogen bond, and the resulting ion pairs, which are related by 21 screw axis along n Fig. 15. Chains et al., 1998abet al., 2000b-glide plane at x = c-glide plane at y = x < 1.0 d Table 1 to form 0 Fig. 16. A secong Fig. 17: the actn − n, n + n + n represents an integer in each case e Fig. 18. Two chaa-glide plane at y = C(7) chains running parallel to the , 2- and 3-chlorobenzoic acids [cf. compound (III)], 2- and 3-bromobenzoic acids [cf. compound (IV)], 2- and 3-iodobenzoic acids, phthalic acid, 3-methylbenzoic acid [cf. compound (I)], 2,4-dichlorobenzoic acid, crotonic and adipic acids [cf. compounds (VIII)–(X)], and ascorbic, aspartic and glutamic acids.All reagents were obtained commercially and were used as received. For the synthesis of each of compounds (I)–(XII), equimolar quantities (0.52 mmol of each component) of U3/U1 ratio was > 4.0: this may be consequence of the disorder. Apart from those in the minor disorder components of (I)–(IV), (VIII)3) or 0.97 Å (CH2), and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups which were permitted to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms: the H atoms bonded to C atoms in the minor disorder components were included on the same basis. The H atoms bonded to O atoms in the disordered components of (VIII)Uiso(H) = 1.5Ueq(O), For the H atoms bonded to N atoms, and for the H atoms bonded to O atoms in (I)–(V), (VII)Uiso(H) = 1.2Ueq(N) or 1.5Ueq(O), leading to the N—H and O—H distances shown in Table 1x parameter . However, for compounds (V)x parameter were indeterminate  global, I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII. DOI: 10.1107/S2056989019012702/ex2024Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019012702/ex2024IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989019012702/ex2024IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989019012702/ex2024IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989019012702/ex2024Vsup6.hklStructure factors: contains datablock(s) V. DOI: 10.1107/S2056989019012702/ex2024VIsup7.hklStructure factors: contains datablock(s) VI. DOI: 10.1107/S2056989019012702/ex2024VIIsup8.hklStructure factors: contains datablock(s) VII. DOI: 10.1107/S2056989019012702/ex2024VIIIsup9.hklStructure factors: contains datablock(s) VIII. DOI: 10.1107/S2056989019012702/ex2024IXsup10.hklStructure factors: contains datablock(s) IX. DOI: 10.1107/S2056989019012702/ex2024Xsup11.hklStructure factors: contains datablock(s) X. DOI: 10.1107/S2056989019012702/ex2024XIsup12.hklStructure factors: contains datablock(s) XI. DOI: 10.1107/S2056989019012702/ex2024XIIsup13.hklStructure factors: contains datablock(s) XII. DOI: Click here for additional data file.10.1107/S2056989019012702/ex2024Isup14.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019012702/ex2024IIsup15.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019012702/ex2024IIIsup16.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019012702/ex2024IVsup17.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019012702/ex2024Vsup18.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019012702/ex2024VIsup19.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019012702/ex2024VIIsup20.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019012702/ex2024XIsup21.cmlSupporting information file. DOI: 1953078, 1953077, 1953076, 1953075, 1953074, 1953073, 1953072, 1953071, 1953070, 1953069, 1953068, 1953067CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Strikingly, AZD8835 promoted robust CD8+ T-cell activation dissociated from its effect on T-regs. This was associated with enhancing effector cell viability/function. Together these data reveal novel mechanisms by which PI3Kα/δ inhibitors interact with the immune system and validate the clinical compound AZD8835 as a novel immunoncology drug, independent of effects on tumor cells. These data support further clinical investigation of PI3K pathway inhibitors as immuno-oncology agents.PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kδ modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. PI3K inhibitors as a class and PI3Kδ specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835, a dual PI3Kα/δ inhibitor, specifically on the tumor immune microenvironment using syngeneic models. Continuous suppression of PI3Kα/δ was not required for anti-tumor activity, as tumor growth inhibition was potentiated by an intermittent dosing/schedule in vivo. Moreover, PI3Kα/δ inhibition delivered strong single agent anti-tumor activity, which was associated with dynamic suppression of T-regs, improved CD8The online version of this article (10.1186/s40425-018-0457-0) contains supplementary material, which is available to authorized users. The phosphoinositide 3-kinase (PI3K) signaling pathway is frequently activated in cancer, promoting tumor cell proliferation and survival. Physiologically this pathway also regulates immune cell function . Whilst + T-cells but also inactivated T-regs cells. This resulted paradoxically in a net gain in anti-tumor immunity in pre-clinical models [PI3K inhibitors with different potencies and isoform selectivities have been developed for different applications. PI3Kα and PI3Kβ isoform inhibitors are classically used as tumor cell targeted agents . The PI3l models . More rel models , 13. Othl models .AZD8835 is a highly selective PI3Kα/δ inhibitor initially developed to treat solid cancers with activating mutations in the PI3K pathway and hence dependent on PI3Kα signaling . HoweverAZD8835 is a potent PI3Kα/δ inhibitor has the potential to inhibit PI3K pathway activity in primary T-cells, which are largely reliant on the PI3Kδ-isoform , 15. PI33) were treated using the same doses and schedules. AZD8835 (PI3Kα/δ) gave greater anti-tumor activity and greater number of tumors responding compared to PI-3065 (PI3Kδ)  Fig. a, c, extKδ) Fig. d-f, despulk Fig. f.Table 1The immune mediated anti-tumor activity of AZD8835 was not restricted to CT26 tumors. AZD8835 promoted anti-tumor immunity in the immunogenic MC-38 colorectal tumor model  Fig. 1gg1g, and + T-cells was sustained with both dosing strategies , then stimulated to activate PI3K signaling. Both AZD8835 and CAL-101 gave dose-dependent reduction of downstream PI3K targets pAkt(Ser473), pS6(Ser244/244) and pNDRG1(T346) by flow cytometry and Western blotting , pS6Ser4/244 andironment , PI3Kα/δ+ T-cell activation, we tested whether AZD8835 or CAL-101 could enhance production of IL-2. AZD8835 promoted a dose-dependent elevation in IL-2 transcript levels  and PI3Kδ (PI-3065) inhibitors are more effective on an intermittent dosing strategy. Moreover, the intermittent dosing strategy revealed that continuous T-regs suppression is not required to deliver robust anti-tumor immune response. Therefore, rather than simply suppressing T-regs function, in the context of the tumor microenvironment PI3K inhibitors enhance or sustain activation of weakly activated CD8 positive cytotoxic T-cells, possibly through IL2 signaling. Importantly the positive effect on the immune system was not context dependent as AZD8835 gave anti-tumor activity in CT-26, 4 T1 and MC-38. All of these models have a unique TME, and response differently to PD1, PDL1 or CTLA4 treatment . Further50 for PI3Kδ for both compounds, adjusted for intrinsic potency for each compound against PI3Kδ isoform. Taken together the data suggests that PI3Kα/δ inhibition is superior because both continuous or intermittent dosing of AZD8835 delivered stronger anti-tumor activity than PI-3065 counterpart groups. Further work will be required to explore optimal profiles using a larger number of PI3K inhibitors with different selectivity profiles.PI3K signaling is critical for tumor progression and enzymes in the pathway are often overexpressed or mutated in a broad range of tumors. As a result, PI3K isoform inhibitors have been tested extensively in the clinic , 6, but It was surprising that acute intermittent inhibition of PI3K signaling is sufficient to drive the positive effects within the tumor immune microenvironment. This contrast with other reports that have only examined sustained PI3Kδ inhibition by genetic ablation or continuous pharmacological dosing , 13, 20.+ T-cell activation phenotypes, treated tumors also contained T-cell memory cells, evidence of increased tumor NK cell accumulation and cytolytic biomarkers and intratumoral IFNγ production.Previous work has concluded that reduction in T-regs as a direct consequence of PI3Kδ inhibition directly contributes to anti-tumor immunity , 12, 20.Based on our in vitro observations we hypothesise that one factor that may contribute to this pro-immune change is that inhibition of PI3K signaling and inhibition of PI3Kδ promotes T-cell function in IL-2 limited conditions, possibly by increasing autocrine IL-2 signaling in effector T-cells. This contrasts with previous studies in knockout mice which suggest that PI3Kδ is critical to mediate T-cell proliferation downstream of TCR signaling . In the Targeting PI3K, AKT or mTORC1/2 may have different effects in specific subsets of tumors , and thiNaïve CD8 T-cells were purified from splenocytes using an EasySep™ Mouse Naïve CD8+ T-cell Isolation Kit (Stem cell Technologies) and cell purification buffer, according to manufacturer’s instructions. T-cells were cultured in RPMI at 2.5-5 × 10^5 cells/mL in a 96-well plate. Cells were activated with 1 μg/mL plate bound αCD3ε (145.2C11) and 1 μg/mL soluble αCD28 (37.51), or with Dynabeads® mouse T-cell activator beads (1:1 bead:cell ratio) at 37 °C in a humidified incubator (5% CO2). Proliferation index refers to the mean number of cell divisions, and was calculated as previously described .To measure growth, 2000 cells/well were seeded in 96-well plates (Costar) and dosed with compounds 24 h later. Cell confluency was monitored at 4 hourly intervals for the duration of the experiment using an Incucyte Zoom platform with 10x objective (Essen Bioscience).All animal studies were performed according to UK Home Office and IACUC guidelines. Cell lines CT-26, 4 T1 and MC-38 we purchased from ATCC. CT-26 (5 × 10^5 cells/mouse) or MC-38 (5 × 10^6 cells/mouse) tumor cells were implanted subcutaneously (s.c.) in the left flank of female Balb/c and C57/Bl6 mice, respectively. 4 T1 (1 × 10^4 cells/mouse) tumor cells were implanted orthotopically in mammy fat pad (o.t.) of female Balb/c mice or sub-cutaneous (s.c). Four days (CT-26 or 4 T1) or one day (MC-38) after implantation mice were randomised by body weight prior to dosing.AZD8835 was dosed at 50 mg/kg twice daily in at 2 days on/ 5 days off schedule for times indicated in figures or 25 mg/kg BID daily . PI-3065At end of study tumor tissues were then transferred into the gentleMACS C Tube containing RPMI. Tumor samples were processed using the mouse tumor dissociation kit from Miltenyi Biotec. Cells were liberated from tumors for downstream application using a mouse tumor dissociation kit and octodissociator (Miltenyi) according to manufacturer’s instructions.The following fluorophore-conjugated antibodies were used in this study: αCD45-Brilliant violet® (BV)786 (30-F11), αCD8α-APC or Brilliant violet® (BV)650 (53–6.7 CD4- Brilliant violet® (BV)711 (RM4–5), NKp46 Brilliant violet® (BV)605 (29A140), αCD11b BUV395 (M1/70), αF4/80-APC (BM8), αCD11c-PE (N418), αMHCII-AF700 (M5/114.15.2), αLy6G-APC-Cy7 (1A8), αLy6C-PerCP/Cy5.5 (HK1.4), αPD-1-Brilliant violet® (BV)421 (29F.1.A12), αGranzyme B-PE (GB12), αCD25-PeCy7 (BC96), Foxp3-APC (FJK-16S), αCD3ε-Brilliant UV® (BUV)395 (17A2) CD69-PE (H1.2F3), CD62L PE-CF594 (MEL14), CD44-BUV737 (IM7), Ki67-Alexa488 (11F6), CD5-FITC (53–7.3), phospho-S6ser240/244)-Alexa647, , phospho-Akt(ser473)-PE (M89–61), Bcl2-PE (clone BCL/10C4), IFNγ-FITC (Clone XMG1.2). All antibodies were purchased from Biolegend, eBioscience, BD or Cell signaling technology. Cells were stained with a viability marker  according to manufacturer’s instructions, and stained for surface/intracellular markers as described previously [0/244-AleCellTrace Violet (CTV) labelling was performed in PBS containing 0.05% BSA and 1.6uM CTV for 10 min at 37 °C. CTV was then quenched following two washes with ice cold MACS buffer. Detection of phospho-specific antibodies was performed following a 25′ stimulation of T-cells at 37 °C. Cells were fixed for 10′ at 37 °C with BD phosflow fixation buffer I, permeabilized for 30′ on ice with pre-chilled (− 20 °C) BD phosflow perm buffer III, and stained for 40′ on ice with BD phosflow perm/wash buffer I, according to manufacturer’s instructions. For detection of intracellular IFNγ, single cell suspensions were derived from tumors and subsequently restimulated with 100 ng/mL PMA, 1μg/mL Ionomycin, 1X Golgi stop (BD) and 1X Golgi plug (BD) in cRPMI , before fixation/permeabilization with a cytofix/cytoperm kit (BD) according to manufacturer’s instructions.Cells were analyzed on a BD fortessa flow cytometer and analyzed using FlowJo software .Detection of IL-2 was carried out using a BD Biosciences OptEIA ELISA kit (BD) according to the manufacturer’s instructions.Total protein lysates were collected in RIPA buffer. Forty micrograms of lysates were run on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were probed with primary antibodies pAkt(Ser473), pS6(Ser240/244), pNDGR1(Thr346) and Vinculin overnight at 4 °C and incubated with secondary antibodies (1:5000 dilution) for 1 h at room temperature. Chemiluminescence was performed with Pierce reagents.Total RNA was isolated from snap frozen tissue and cells using Qiashredder and Qiazol Lysis Buffer on Qiacube-HT following the RNeasy 96 QIAcube HT total RNA cell with DNase protocol according to manufacturer’s instructions (Qiagen). Reverse transcription was performed from 50 ng of total RNA (Thermo Scientific #4374967) and genes of interest were pre-amplified  using a pool of TaqMan primers  was used to quality control and analyse the sequencing data. Within bcbio, the sequencing reads were aligned using hisat2 2.1.0 for quality control purposes and a QC report was generated using multiqc Quantification of expression of the transcripts  was performed directly against the mouse mm10 Ensembl transcriptome using Salmon 0.9.1 [p-values obtained by DESeq2. A customised support vector regression (SVR) model was developed in-house based on the CIBERSORT algorithm to achieve immune cell deconvolution [www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-7386.For RNA sequencing, total RNA was extracted using the RNeasy 96 Qiacube HT Kit (Qiagen), quality validated using nanodrop and Quantit RNA Assay Kit (Thermo Fisher), and submitted for TrueSeq Stranded mRNA library preparation, following the manufacturer’s instructions (Illumina). Resulting libraries were sequenced on the HiSeq4000 System. The python toolkit bcbio 1.0.8  and indicated in the legend. Statistical significance is indicated as follows: * Additional file 1:Figure S1. Inhibition of PI3Kα/δ improves overall survival in CT-26 tumor and is active in low and high immunogenic immunocompetent models MC-38 and 4 T1. (A) Kaplan-Meyer curve shows survival fractions in CT-26 tumor bearing mice treated at indicated doses with AZD8835 at intermittent or continuous schedule  and intermittent and continuous treatment with PI-3065 . Data are representative of ≥2 independent experiments. (PDF 60 kb)Additional file 2:Table S1. Immuno-phenotyping antibodies used for tumor flow cytometry. (DOCX 13 kb)Additional file 3:Figure S2. Low dose of PI3Kα/δ inhibitor suppresses tumor T-regs independent of efficacy. (A) Line graph shows mean tumor volumes from BALB/c mice bearing CT-26 tumors dosed 4 days after cell implant at indicated doses. (B) Line graph shows individual tumor volumes from BALB/c mice bearing CT-26 tumors. Grey area in plot indicates continuous schedule and dashed lines indicate 2 days on/5 days off intermittent schedules at indicated doses of AZD8835 or PI-3065. (C) Scatter plots represent relative tumor T-regs cell frequencies relative to CD45+ cells. (D) Scatter plots represent tumor CD8/T-regs ratios. (PDF 86 kb)Additional file 4:Figure S3. Immune phenotyping of MC-38 tumors treated with AZD8835. Scatter plot shows relative quantification of (A) cytotoxic CD8+ T-cells, (B) Mo-MDSCs, (C) DCs, (D) Macrophages, (E) G-MDSC/Neutrophil and (F) NK cells of treated and untreated tumors with AZD8835 (PI3Kα/δ) 50 mg/kg 2on/5off for a period of 10 days. Error bars represent mean ± SEM, statistical differences were calculated using a 1-way ANOVA with post hoc analysis. Data are representative of 2 independent experiments. Statistical significance is indicated as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (PDF 77 kb)Additional file 5:Table S2. List of differentiated expressed genes in control and AZD8835 treated tumors at day 7 and day 14. (XLXS 59 kb)Additional file 6:Figure S4. Direct target engagement in primary immune cells. (A-B) CD8+ T-cells were purified from spleens, preincubated with inhibitors AZD8835 (α/δ) AZD8186 (β/δ) and CAL101 (δ) for 1 h, then stimulated with 10 μg/mL α-CD3 and 2 μg/mL soluble α-CD28 for 25 min at 37 °C. (A-B) Line graph shows MFI of pAkt(Ser473) and pS6(Ser240/244), histograms show representative data. Data are representative of ≥2 independent experiments. (C) Cell lysates were prepared, separated by SDS-PAGE, and immunoblotted to detect pS6(Ser240/244), pNDRG-1(Thr346) and Vinculin. (PDF 307 kb)Additional file 7:Figure S5. IL2 RNA and Bcl family RNA heatmap and protein levels. (A) Purified naïve CD8+ T-cells were rested for 3 days with AZD8835 at a maximum of 10 μM under stimulated conditions with CD3/CD28 beads. (B) Heatmap shows mRNA expression levels of anti-apoptotic factors on purified CD8+ T-cells treated as in A. (C) Histogram showing increased mean fluorescence intensity (MFI) levels of BCL2 protein in cell treated with 0.3 μM of AZD8835 and CAL-101. Data represent ≥2 experiments. (PDF 75 kb)Additional file 8:Table S3. TaqMan primers panel of 96 genes used to calculate a GSVA score. (DOCX 17 kb)


The crystal structures of two aromatic amines are presented: a hydrogen-bonded brominated aniline, and a piperizine derivative. N-bromosuccinimide affords the ortho-brominated derivative 2-bromo-4,6-dimethylaniline , which sublimates in vacuo to afford crystals featuring hydrogen-bonded chains as well as Type I halogen–halogen interactions. Conversely, alkylation of two equivalents of 2,4-dimethylaniline with 1,2-dibromoethane affords a separable mixture of N,N′-bispiperazine , which was crystallographically characterized, as well as N,N′-bisethylenediamine (3).Crystallographic studies of nitrogen-containing small molecules aid in the elucidation of their structure–activity relationships and modes of aggregation. In this study, two previously synthesized molecules are crystallographically characterized for the first time. Reaction of 2,4-dimethylaniline with  Halogenated NHCs can be utilized to fine-tune the steric and electronic properties of transition metal catalysts. It has been demonstated that the presence of fluorine on an aryl group of an NHC ligand influences the E/Z selectivity of a ruthenium cross-metathesis catalyst  and N,N′-bispiperazine (2). Though available from many commercial suppliers, the crystal structure of 2-bromo-4,6-dimethylaniline (1) has not been previously disclosed. Only a few reports of compound 2 can be found in the literature. An early publication ethanol via palladium-mediated hydrogen autotransfer between 2,4-dimethylaniline and ethylene glycol  = 3.172 (5) Å] within the P21/c space group. The two independent molecules exhibit C—Br and C—N bond lengths that are equal within experimental error . The angle between the two mean planes passing through the aromatic rings of the two independent molecules is 80.6 (2)°. The hydrogen atoms on each nitrogen centre that are not involved in the chains formed by the N—H⋯N interactions are oriented towards the ortho bromine atoms on the aromatic rings. These intramolecular hydrogen bonds feature donor-acceptor distances of 3.082 (4) and 3.094 (4) Å.The solid-state structure of 2 revealed a solvent-free structure in the P21 space group  and 56.9 (1)° for C5–C10 and C13–C18, respectively.X-ray diffraction analysis of up Fig. 2. The asy1 provides one donor and one acceptor site for the hydrogen-bond interactions , this classifies as a Type I halogen–halogen interaction  Å] and a weaker bromine⋯bromine interaction (Br⋯Br distance of 3.637 (1) Å), both observed along the 31 screw axes, and with additional intramolecular N–H⋯Br interactions. In the case of the more sterically hindered derivative 1, this arrangement is not feasible and chains are instead adopted.The packing motif of compound et al., 20122 are twisted relative to the N2C4 mean plane, the structure of the less substituted N,N′-diphenylpiperazine, which crystallizes in the Pbca space group, exhibits phenyl groups closer to being in conjugation with the nitrogen lone pairs , though the N–Caryl bond lengths are slightly shortened in the phenyl-substituted analogue , indicating that resonance delocalization is a perhaps a minor effect, if present, while packing effects likely dominate. The structures of 2 and the phenyl analogue are overlaid in Fig. 6Most of the crystallographically characterized diarylpiperazines feature the chair conformation; a few have been determined in the twist-boat form ethylenediamine (3) via a condensation reaction. Compound 3 is evidently able to compete with 2,4-dimethylaniline as a nucleophile towards 1,2-dibromoethane, once formed. Both desired main product 3 and by-product 2 were isolated after separation by column chromatography.The piperazine compound 1H and 13C resonances are referenced to residual CHCl3 or CDCl3, respectively, using the reported values relative to SiMe4 , ammonium acetate , and acetonitrile (60 mL). The reagent 2,4-dimethylaniline  was added slowly, by pipette. The resulting mixture was left to stir at room temperature for 90 min. The solvent was removed under vacuum to produce a reddish-brown solid. Water (45 mL) and dichloromethane (45 mL) were added, and the mixture was transferred to a separatory funnel. The organic layer was separated and washed with water (3 × 30 mL), saturated sodium thiosulfate (30 mL), and brine (30 mL). After drying the organic layer with magnesium sulfate, the mixture was filtered and the volatiles removed under vacuum to afford a brown crystalline solid . 1H NMR : δ 7.15 , 7.14 , 3.93 , 2.22 , 2.21 . The procedure was based on one reported for similar aniline derivatives . The product can be purified by sublimation under static vacuum with heating to 308 K for 3 d. Large X-ray quality crystals of the product were obtained by slow sublimation under ambient conditions in a capped glass vial containing the crude product, over a period of months.A 100 mL round-bottom flask equipped with a magnetic stir bar was charged with N,N′-diisopropylethylamine , and fitted with a reflux condenser and drying tube. The mixture was heated to 403 K for 4 h, then cooled to room temperature affording a red solid mass. To this was added H2O (50 mL) before extraction with CH2Cl2 (30 mL). The organic phase was washed with H2O (50 mL), and to the combined aqueous extracts was added 1 M NaOH(aq) (40 mL), and this mixture was extracted with CH2Cl2 (50 mL). The combined organic extracts were washed with H2O (30 mL) and brine (30 mL), dried over MgSO4, decanted into a round-bottom flask, and dried under vacuum to afford a dark orange–red liquid. Addition of hexanes (40 mL) resulted in the precipitation of crystalline material. The solid material was redissolved by warming the hexanes, and the resultant clear red solution was stored overnight at 238 K. The mother liquor was decanted and the remaining solid material was washed with cold hexanes (3 × 3 mL) and dried under vacuum to afford a beige solid (5.4730 g). NMR data indicated that the product was a 90:10 mol% mixture of 1,2-diamine 3 and piperazine 2, obtained with a 69% yield of products based on 1,2-dibromoethane. Separation of the compounds was achieved by silica gel flash chromatography. Elution of 1.3223 g of a mixture with CH2Cl2 afforded piperazine 2 as a pale-tan crystalline solid  and diamine 3 as a pale-yellow solid . The 1H and 13C chemical shifts and assignments for diamine in CDCl3 differed from the reported values : δ 6.96 , 6.91 , 6.61 , 3.61 , 3.47 , 2.25 , 2.11 . 13C{1H} NMR (CDCl3): δ 144.0 (s), 131.3 (s), 127.6 (s), 126.8 (s), 122.8 (s), 110.4 (s), 43.8 , 20.5 (s), 17.7 (s). Piperazine 2: 1H NMR (CDCl3): δ 7.03 , 7.01 , 3.04 , 2.33 , 2.30 . 13C{1H} NMR (CDCl3): δ 149.5 (s), 132.9 (s), 132.8 (s), 132.0 (s), 127.2 (s), 119.3 (s), 52.8 (s), 20.9 (s), 18.0 (s). The procedure was based on that used for the fluoro analogue , 1,2-dibromoethane , and 1 were located in the difference map and refined freely. The piperazine 2 crystallized in the non-centric group P21; no heavy atoms are present in the structure, therefore the Flack parameter was not calculated. Carbon-bound hydrogen atoms were placed in calculated positions (C—H = 0.95–0.99 Å) and refined according to a riding model, with fixed Uiso values of 1.2 times (CH and CH2 groups) and 1.5 times (CH3 groups) the parent atom.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018011404/lh5879sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989018011404/lh58791sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989018011404/lh58792sup3.hklStructure factors: contains datablock(s) 2. DOI: 1861254, 1861253CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The planar and relative structures of 1–3 were established via high-resolution electrospray ionisation mass spectrometry and one- and two-dimensional nuclear magnetic resonance spectroscopic analyses, and the absolute configuration of 1 was further established via X-ray crystallography experiment. Compounds 1–3 showed selective inhibitory potency against certain human tumor cell lines with IC50 values ranging from 8.50 ± 0.13 to 35.52 ± 0.08 μM.Three new The online version of this article (10.1007/s12272-019-01151-y) contains supplementary material, which is available to authorized users. Euphorbia fischeriana Steud, a perennial herbaceous plant of the Euphorbiaceae family, is widely distributed in northeastern mainland China . Here, we report the isolation, structural elucidation, and cytotoxic activity of these compounds  as the internal standard. The chemical shifts were reported in δ (ppm) units relative to the TMS signal and coupling constants (J) in Hz. High-resolution electrospray ionisation mass spectrometry (HRESIMS) data were collected in the positive ion mode on the Thermo Fisher LC-LTQ-Orbitrap XL spectrometer. Column chromatography separations were performed on a silica gel , ODS column , and Sephadex LH-20 liquid chromatography media . Semi-preparative high-performance liquid chromatography (HPLC) analysis was performed with the Agilent 1100 quaternary system using the reversed-phased C18 column . Thin layer chromatography was carried out with pre-coated silica gel 60 F254  plates, and spots were visualized by spraying heated silica gel plates with 10% sulfuric acid in ethanol.Optical rotations were collected on the JASCO P2000 automatic digital polarimeter. Ultraviolet visible spectra were collected on a Hewlett-Packard HP-845 UV–VIS spectrophotometer. FT-IR spectra were recorded on the Bruker Vertex 70 instrument. 1D and 2D nuclear magnetic resonance (NMR) spectra were measured on the Bruker AM-400 spectrometer, with pyridine-E. fischeriana were collected from the Wudang Mountain area of Hubei Province of China in September 2016. The voucher specimen (20160925) was deposited in Hubei University of Medicine .The roots of E. fischeriana (4.0 kg) were chopped and extracted with 80% acetone (3 × 5 L) at ambient temperature (298 K). The combined extracts were concentrated under reduced pressure to yield a crude residue (140 g), which was suspended in water (2.0 L) followed by successive partitions with ethyl acetate (EtOAc) (3 × 1.5 L) and n-BuOH (3 × 2.0 L). The EtOAc extracts (45 g) were subjected to a silica gel column chromatography eluted with CH2Cl2–acetone  to afford six fractions, Fr.1–Fr.6. Then Fr.3 (12 g) was loaded onto an RP-C18 column with stepwise MeOH–H2O  to yield five fractions (Fr.3.1–Fr.3.5). Fr.3.2 (3.3 g) was purified by silica gel column chromatography  and repeated semi-preparative HPLC analysis using MeOH–H2O  as eluent to provide compound 3 . Fr.4 (15 g) was purified by an RP-C18 CC using MeOH–H2O , a silica gel CC eluted with CH2Cl2–MeOH , and repeated semi-preparative HPLC using MeOH–H2O  to afford compounds 1  and 2 (8.4 mg tR = 22 min).The air-dried roots of α]23 D: +13.5 ; UV (MeOH) λmax (log ε): 204 (4.18) nm; IR (νmax): 3429, 1693, 1628, 1551, 1384, 1055, 584 cm−1; for 1H- and 13C-NMR spectroscopic data, see Table m/z 333.2070 .Colorless crystals; [α]23 D: –15.2 ; UV (MeOH) λmax (log ε): 203 (4.12) nm; IR (νmax): 3418, 2934, 1716, 1630, 1383, 1221, 1056, and 655 cm−1; for 1H- and 13C-NMR spectroscopic data, see Table m/z 331.1915 .White powders; [α]23 D: +25.8 ; UV (MeOH) λmax (log ε): 205 (3.89) nm; IR (νmax): 3421, 2924, 2854, 1677, 1619, 1442, 1381, 1259, 1207, 1156, 1100, 1034, and 864 cm−1; for 1H- and 13C-NMR spectroscopic data, see Table m/z 373.2380 .White powders; .Crystals of 1: C20H30O4, M = 334.44, monoclinic, a = 10.4886(5) Å, b = 7.3402(4) Å, c = 11.3529(6) Å, α = 90.00°, β = 99.013(2)°, γ = 90.00°, V = 863.25(8) Å3, T = 100(2) K, space group P21, Z = 2, μ(Cu Kα) = 0.703 mm−1, 5155 reflections measured, 2536 independent reflections (Rint = 0.0595). The final R1 values were 0.1336 (I > 2σ(I)). The final wR(F2) values were 0.3012 (I > 2σ(I)). The final R1 values were 0.1337 . The final wR(F2) values were 0.3016 . The goodness of fit on F2 was 1.499. Flack parameter = 0.1(4).Crystallographic data for compound 2. The MTT assay was conducted to measure cell survival. In brief, a suspension (100 μL) of adherent cells was seeded into 96-well culture plates and allowed to adhere for 12 h before addition of the tested compounds. The suspended cells were seeded at a density of 1 × 105 cells/mL immediately before drug additions. Each tumor cell line was exposed for 48 h in triplicate to the tested compounds at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM, and cisplatin  was selected as the positive control. After incubation, culture supernatants were removed and exchanged with medium containing MTT (0.5 mg/mL). Then the cells were incubated at 37 °C for 4 h in the dark, followed by the removal of the medium and addition of dimethyl sulfoxide (100 μL). The absorbance at 570 nm was measured, and the data were expressed as the average of three replicates of absorbance in treated versus control cells.Six human tumor cell lines, HL-60, SMMC-7721, MM-231, A-549, HEP3B, and SW-480, along with one noncancerous cell line, the human normal colonic epithelial cell NCM460, were used in the cytotoxicity assay  was obtained as white powders, and had a molecular formula of C20H30O4 based on its HRESIMS data at m/z 333.2070 , corresponding to six degrees of unsaturation. Detailed interpretation of the 1H- and 13C- NMR and HSQC spectra disclosed the obvious signals assigned as follows: two singlet methyls , eight methylenes , five methines , and five quaternary carbons . Among them, two double bonds and one carboxyl group occupied three of six degrees of unsaturation, suggesting that this compound had a tricyclic ring system.Fischerianoid A -diene-3β,16,18-triol /H-13 indicated that H-13, Me-19, and Me-20 were all α-oriented, on the contrary, H-3, H-5, H-9, and CH2(OH)-18 were all β-oriented. Accordingly, the relative configuration of 1 was defined, which was consistent with that of ent-abieta-7,15(17)-diene-3β,16,18-triol. To further determine its absolute configuration, the crystallographic approach was considered. Fortunately, after repeated recrystallization by various two-phase or three-phase solvents, a suitable crystal of 1 was obtained from methanol/chloroform/water (4:4:1) at room temperature, which was then analyzed by a single-crystal X-ray diffraction experiment using Cu Kα  was obtained as white powders, and possessed a molecular formula of C20H28O4 based on the HRESIMS analysis at m/z 331.1915 , which was two hydrogen atoms less than 1. Detailed comparison of the 1D NMR data  replaced one oxygenated methylene group at δC 67.7 . This deduction was further confirmed by the HMBC correlations  was deduced from the HRESIMS data at m/z 373.2380  and 13C NMR data, which corresponded to seven degrees of unsaturation. A side-by-side comparison of the 1D NMR data , CH3-3′ (δH 1.55/δC 30.6), and C-1′ (δC 99.4)] were present, suggesting that 3 was an acetonide derivative of 1. This conclusion was further supported by one more degree of unsaturation required in the molecule of 3 and the HMBC correlations , were isolated and identified from the EtOAc extracts of the roots of E. fischeriana. The planar and relative structures of 1–3 were established via extensive spectroscopic data (HRESIMS and 1D and 2D NMR) analyses, and the absolute configuration of 1 was further established via X-ray crystallography experiment with Cu Kα radiation. Compound 3 was characterized by a rare acetonide motif but most possibly an artifact of compound 1 generated during the extraction process. Additionally, compounds 1–3 showed selective inhibitory potency against certain human tumor cell lines with IC50 values ranging from 8.50 ± 0.13 to 35.52 ± 0.08 μM, with no obvious cytotoxicity to the normal cell NCM460. These findings not only enrich the new numbers of ent-abietane diterpenoids, but also extend the pharmaceutical usage of ent-abietane diterpenoids to cytotoxic activity. More importantly, they may play important roles in the chemotaxonomic significance of E. fischeriana as well as the genus Euphorbia.Previous phytochemical investigations on Supplementary material 1 (DOC 1380 kb)Below is the link to the electronic supplementary material.


Protein data is available at Proteomics Identifications (PRIDE) partner repository (PRIDE identifier PXD012134).A growth cone is a part of a neuron considered as a hub for axon growth, motility and guidance functions. Growth cones are thought to play a critical role during development of neurons. Growth cones also play a significant role in adult regeneration. Here, we present a dataset on the lipid and protein profiling of the growth cone-enriched fractions derived from C57BL/6J mice forebrains of developmental stage: E18, P0, P3, P6, and P9. For comparison, we analyzed non-growth cone membranes from the same samples. Lipid data is available at the Metabolomics Workbench [ Lipids were extracted using chloroform, methanol and water mixture to obtain phase separation. We then used untargeted liquid chromatography Q-Exactive Orbitrap tandem mass spectrometry (LC-MS/MS) for lipid profiling. Peak extraction, identification, relative quantification, and alignment were performed using Lipid Search 4.1 software. The list of identified lipids is provided in 22.1All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and policies of the UM Institutional Animal Care & Use Committee (IACUC). Forebrains of E18, P0, P3, P6, and P9 C57BL6/J mice were fractionated by sucrose gradient ultracentrifugation into growth cone-enriched (GC) and non-growth cone membrane (M) fractions as described previously 2.22.2.16 mL of methanol (LC-MS grade) and 3 mL of chloroform (LC-MS grade) were added to each sample . After 2 min vigorous vortexing and 2 min sonication in an ultrasonic bath, the samples were incubated at 48 °C overnight . The following day, 3 mL of water (LC-MS grade) and 1.5 mL of chloroform were added, samples vigorously vortexed for 2 min and centrifuged at 3000 RCF, 4 °C for 15 min to obtain phase separation. Lower phases were collected. Remaining samples (upper and interphases) were re-extracted by addition of 4.5 mL of chloroform and centrifugation at 3000 RCF, 4 °C for 15 min to obtain phase separation. Lower phases from both extractions were combined and dried in a centrifugal vacuum concentrator. Samples were stored at −20 °C until reconstituted in 150 μL of chloroform:methanol (1:1) before mass spectrometric analysis.2.2.1.1Reversed-phase chromatographic separation utilized Accela Autosampler (Thermo), Accela 600 pump (Thermo) and Acclaim C30 column: 3 μm, 2.1 × 150 mm (Thermo). The column temperature was maintained at 30 °C and tray temperature at 20 °C. Solvent A was composed of 10 mM ammonium acetate (LC-MS grade) in 60:40 methanol:water (LC-MS grade) with 0.2% formic acid . Solvent B was composed of 10 mM ammonium acetate with 60:40 methanol:chloroform with 0.2% FA. The flow rate was 260 μL/min, and the injection volume was 10 μL. The gradient was 35–100% solvent B over 13.0  min, 100% solvent B over 13.0–13.8 min, 100–35% solvent B over 13.8–14.5 min, 35% solvent B over 14.5–18.0 min, 0% solvent B over 18.0–20.0 min.2.2.26 ions and maximum ion injection time (IT) of 100 ms. Data-dependent MS/MS (top10) were acquired using the following parameters: resolution 17,500; AGC 1 × 105; maximum IT 75 ms; 1.3 m/z isolation window; underfill ratio 0.1%; intensity threshold 1 × 103; dynamic exclusion time 3 s. Normalized collision energy (NCE) settings were: 15, 30, 45, 60, 75, 90 .The Q Exactive (Thermo) mass spectrometer was operated under heated electrospray ionization (HESI) in positive and negative mode separately. The spray voltage was 4.4 kV, the heated capillary was held at 310 °C (negative mode) or 350 °C (positive mode) and heater at 275 °C (positive mode). The S-lens radio frequency (RF) level was 70. The sheath gas flow rate was 30 (negative mode) or 45 units (positive mode), and auxiliary gas was 14 (negative mode) or 15 units (positive mode). Full scan used resolution 70,000 with automatic gain control (AGC) target of 1 × 102.2.2.1Lipid identification and relative quantification were performed using LipidSearch 4.1 software (Thermo). The search criteria were as follows: product search; parent m/z tolerance 5 ppm; product m/z tolerance 5 ppm; quantification: m/z tolerance 5 ppm, retention time tolerance 1 min. The following adducts were allowed in positive mode: +H, +NH4, +H—H2O, +H—2H2O, +2H, +Na, +K and negative mode: H, +HCOO, +CH3COO, -2H, —Cl. All classes were selected for search. LipidSearch nomenclature is used .Table 2L2.2.3Positive and negative mode identifications at different NCE were aligned in LipidSearch, allowing calculation of unassigned peaks. The following settings were applied: product search; alignment method max; retention time tolerance 0.1 min; filters: top rank, main isomer peak; M-score 5; molecular lipid identification grade: A-B (A: lipid class and fatty acid completely identified or B: lipid class and some fatty acid identified).2.32.3.1100 μg of proteins were precipitated with 4 vol of ice-cold acetone overnight at −20 °C. The following day, proteins were pelleted by centrifugation . Pellets were resuspended in 20 μL of 0.2% ProteaseMAX (Promega)/50 mM ammonium bicarbonate (ABC). 50 mM ABC was added to a final volume of 93.5 μL. Proteins were reduced with dithiothreitol (DTT): 1 μL of 0.5 M  for 20 min at 56 °C. Next, proteins were alkylated using iodoacetamide (IAA): 2.7 μL of 0.55 M  for 15 min at room temperature in the dark. 1 μL of 1% ProteaseMAX/50 mM ABC with 4 μg of Trypsin/Lys-C mix  was added. Digestion was carried out in a total volume of 100 μL  for 4 h at 37 °C. Samples were adjusted to 300 μL of 0.1% trifluoroacetic acid/50 mM ABC. Pierce High pH Reversed-Phase Fractionation Kit (Thermo) was used to separate peptides into eight fractions (according to manufacturer's instruction). We also collected flow-through, wash (water) and an additional 100% acetonitrile (ACN) elution fraction . Fractions were dried in a centrifugal vacuum concentrator at 45 °C and then stored at −20 °C. Before LC-MS analysis, peptides were re-suspended in 30 μL of 0.1% FA in water.2.3.2The chromatographic separation utilized EASY-nLC 1000 system (Thermo) and Acclaim PepMap RSLC 75 μm × 15 cm, nanoViper column (Thermo). Solvent A was composed of 0.1% FA in water (LC-MS grade). Solvent B was composed of 0.1% FA in ACN (LC-MS grade). The flow rate was 260 μL/min and injection volume was 10 μL. The gradient was 2–5% solvent B over 5.0  min, 5–60% solvent B over 5–70  min, 60–98% solvent B over 70–98  min, 98% solvent B over 98–118 min, 98-2% solvent B over 118–120 min.2.3.36 ions and maximum ion injection time (IT) of 100 ms. Data-dependent MS/MS (top15) were acquired using the following parameters: resolution 17,500; AGC 1 × 105; maximum IT 200 ms; 1.3 m/z isolation window; NCE 28. Underfill ratio was set to 0.1%; intensity threshold to 5 × 102; dynamic exclusion time to 20 s; peptide match to preferred; charge exclusion to unassigned and 1.The Q Exactive (Thermo) mass spectrometer was operated under heated electrospray ionization (HESI) in positive mode. The spray voltage was 1.8 kV, the heated capillary was held at 250 °C, and the S-lens radio frequency (RF) level was 70. Full scan (m/z 150–2000) used resolution 70,000 with automatic gain control (AGC) target of 1 × 102.3.4The acquired raw files were analyzed with Proteome Discoverer 2.2 (Thermo) using the SEQUEST HT engine. The data was searched against 83,916 Mus musculus entries . Search parameters included: precursor mass tolerance 10 ppm and 0.02 Da for fragments, 2 missed trypsin cleavages, oxidation (Met) and acetylation (protein N-term) as variable modifications, carbamidomethylation (Cys) as a static modification. Percolator PSM validation used the following parameters: strict FDR of 0.01, relaxed FDR of 0.1, maximum ΔCn of 0.05, validation based on q-value. For label-free quantification (LFQ), the Minora Feature Detector was used along with the Feature Mapper and Precursor Ions Quantifier. Data were filtered by peptide filter: medium (FDR 0.1) and high confidence (FDR 0.01) and protein filter: medium (FDR 0.1) and high confidence (FDR 0.01).