Bacillus subtilis (B. subtilis) BJ3-2 strain has various metabolic properties at different temperatures, and the strain cultured at 37℃ increased the soybean flavor  compared with culturing at 45℃ and 53℃. Interestingly, the strain cultured at 45℃ and 53℃ had a higher soy sauce-like flavor than that in 37℃. Moreover, a comparative transcriptome analysis of the strain cultured at 37℃, 45℃, and 53℃ showed transcriptional changes related to secondary metabolites and ABC transporters, which is critical for the amino acid transport and metabolism in B. subtilis. Meanwhile, proteomics and metabolomics profiling showed a marked change in amino acids transport and metabolism. In addition, the metabolic analysis revealed a significant metabolic difference  in the strain cultured at 45℃ and 53℃ compared to 37℃. To sum, this study used the multi-omics profiling tool to investigate the fermentative strains B. subtilis BJ3-2, thus providing a deeper insight into the mechanism of the formation of soy sauce-like flavor and soybean flavor compounds.Although soy sauce-like flavor and soybean flavor are two key contributors to the flavor of fermented foods, the key compounds of soy sauce-like flavor and soybean flavor and production mechanisms are still poorly understood and need further investigation. In the present study, we found that the The online version contains supplementary material available at 10.1186/s12866-022-02555-5. B. subtilis is one of the most dominant microorganisms found in fermented foods [B. subtilis is an inexpensive and effective process that improves nourishing and functional food ingredients [B. subtilis can produce a variety of components during fermented processes, such as protease, amylase, and lipase, all of which have important roles in the hydrolysis of proteins, starches, and lipids [B. subtilis significantly contribute to the formation of flavor compounds during the Baijiu brewing process [B. subtilis has been acknowledged as an important bacterium for forming flavor compounds in fermented foods [The unique flavor and nutritional value of the fermented foods result from numerous microorganisms and their metabolites produced during fermentation , 2. The ed foods , 6. Fermredients , 8. For d lipids . In addi process . Therefoed foods , 9, 11.B. subtilis have been widely reported as greatly impacting the flavor of fermented foods and other foods [B. subtilis). Homoplastically, natto is a traditional Japanese food obtained after fermentation of the soybeans by B. subtilis [Maotai-flavor of Chinese liquors [B. subtilis grown in solid substrate conditions using soybean suspended in water and supplemented with threonine and acetoin [Among all the flavor chemicals, acetoin and ligustrazine, produced by er foods –15. Tetrsubtilis , 17. How liquors . Moreove acetoin  as precu acetoin , 21. It  acetoin . Moreove acetoin , 24. Yet acetoin .Bacillus licheniformis isolated from the Maotai flavor liquor produced a strong soy sauce-like flavor in the medium of wheat bran extract by submerged fermentation [Recently, a dominant strain of entation . More imentation . Additioentation . Moreoveentation ; howeverB. subtilis BJ3-2 were isolated and identified from the bacteria-fermented lobster sauce samples which collected from 10 producing areas in Guizhou province [In our previous study, a strain of province . In thisBacillus subtilis BJ3-2 as previously described [The culture and collection of escribed , 30. BriP-value < 0.05 and |log2fold change (FC) |≥ 1. Gene Ontology  enrichment analysis was performed to determine the DEGs that were common at 37 °C, 45 °C, and 53 °C with opposite regulatory patterns with a threshold of P < 0.05. Kyoto Encyclopedia of Genes and Genomes  pathway enrichment analysis was performed for each DEG [The total RNA was extracted using TRIzol reagent  by following the manufacturer's protocol. The concentration and purity of the total RNA were evaluated using a Nanodrop ND-2000 spectrophotometer . The RNA integrity values of the total RNA were determined using an Agilent 2100 bioanalyzer . Each sample was collected at least three times and pooled for RNA sequencing (RNA-seq) analysis. RNA-seq was performed by the Majorbio Technology Co., Ltd. . The 16S rRNA was used as an internal standard. Sequences of the raw data containing a small number of reads with sequencing adaptors or low-quality sequences were filtered out. Subsequently, the quality of sequencing data was evaluated by a sequencing error rate distribution examination. For gene expression analysis, the reads counts were normalized to Fragments Per Kilo bases per Million fragments (FPKM), and the differentially expressed genes (DEGs) were selected using the following conditions: each DEG .2O with 0.1% FA, 2% ACN (phase A) and 80% ACN, 0.1% FA (phase B). Separation of the sample was performed with a 90 min gradient at 300 nL/min flow rate; Gradient B: 2–5% for 2 min, 5–22% for 68 min, 22–45% for 16 min, 45–95% for 2 min, 95% for 2 min. The data acquisition was performed with the data-dependent acquisition (DDA) in profile and the DDA of MS1 spectra was performed in positive mode with an Orbitrap analyzer at a resolution of 120,000 (at m/z of 200) and 350–1600 m/z range. The MS2, resolution was set at 45 k and fixed first mass was set to 100 m/z. The automatic gain control target was set to 3E6 (with the max IT 30 ms) and 1E5 (with the max IT 96 ms) in MS1 and MS2, respectively. The top 20 most intense ions were fragmented by high-energy dissociation (HCD) with normalized collision energy of 32% and isolation window of 0.7 m/z. The dynamic exclusion was used with a time window of 45 s. The single charged peaks and peaks with charges exceeding 6 were masked and excluded from further analyses.For each sample, 2 μg of total peptides were separated and analyzed with a nano-UPLC (EASY-nLC1200) coupled to a Q Exactive HFX Orbitrap instrument (Thermo Fisher Scientific) with a nano-electrospray ion source according to the method of Liu with some modifications . SeparatThe vendor’s raw MS files were processed using Proteome Discoverer (PD) software (Version 2.4.0.305) and the built-in Sequest HT search engine according to the description with some modifications . MS specThe metabolites extraction according to the methods described with minor modifications , 34. Bri−1and the gas flow rate through the column was 1 mL min−1. The column temperature was programmed as follow: the initial temperature at 90 °C (held for 1 min), increased to 180 °C at 10 °C min−1 (held for 1 min), increased to 240 °C at 5 °C min−1 (held on 1 min), increased to 300 °C at 30 °C min−1 (held on 12 min). The injection, transfer line and ion source temperatures were set at 280, 250, and 220 °C respectively. The ionisation mode was operated in electron impact (electron energy -70 eV). The mass spectrometry data were acquired in the full-scan mode at a rate of 100 spectra per second using a mass scan range of 33–550 m/z after a solvent delay of 5.85 min.Agilent 7890 gas chromatograph system coupled with a Pegasus 4D time of flight mass spectrometer was used for GC × GC-TOF–MS analysis according to the methods described by previous report with minor modifications –35. A 1 P-value < 0.05 and |log2fold change (FC) |≥ 1.Chroma TOF software of LECO Corporation and Fiehn database were used for raw peaks exacting, the data baselines filtering and calibration of the baseline, peak alignment, deconvolution analysis, peak identification, and integration of the peak area . Next, tBacillus licheniformis CGMCC3962 [Bacillus subtilis BJ3-2 indicated that the metabolic regulation was not constant at the various temperatures.In the present study, BJ3-2 was selected as the target strain, whose growth and physiological characterization was investigated by culturing in LB medium at different temperatures. The results showed that the strain cultured at 53℃ had a darker color compared to that cultured at 37℃, while only a slight color difference was observed at 45℃ Fig. SA. MoreovGMCC3962 . ConsequP-value < 0.05 and |log2FC|≥ 1 as the significance. As shown, 841 DEGs were identified in BT45 vs. AT37; among them, there were 521 down-regulated DEGs and 320 up-regulated DEGs. In comparison with the DEGs of AT37 and CT53, 1970 DEGs were identified; among them, there were 1063 down-regulated DEGs and 907 up-regulated DEGs. Similarly, 1043 DEGs were identified in CT53 vs. BT45; among them, there were 576 down-regulated DEGs and 467 up-regulated DEGs , including 23 885 294 raw reads from AT37 samples, 24 158 336 raw reads from AT45 samples, and 23 125 998 raw reads from AT53 samples. The raw sequences were filtered, and 23 738 006, 24 012 568, and 22 974 490 clean reads were generated for AT37, BT45, and CT53, respectively , molecular function (MF), and cellular component (CC). All the DEGs in BT45 vs. AT37 were dominantly categorized into the MF categories, and most of the DEGs were connected to serine-type peptidase activity and serine hydrolase activity Fig. , 7. HoweP-value < 0.05 and |log2FC|≥ 1). As shown, 72 down-regulated and 195 up-regulated proteins were identified in BP45 vs. AP37. Similarly, 132 down-regulated and 224 up-regulated proteins were found when comparing AP37 with CP53. Likewise, 224 down-regulated and 125 up-regulated proteins were identified in CP53 vs. BP45  analysis Fig. . InteresFig. A. HoweveFig. B, C. MeaFig. A. SimilaFig. B. NotablFig. C, which To further compare the metabolite compositions in A37, B45, and C53, the samples of these strains were subjected to GC × GC-TOF–MS analysis. The score scatter plot for the PCA model indicated that the samples were clearly isolated within the PC1 × PC2 score plots Fig. A. The voB. subtilis is an important microorganism widely used in the food industry [B. subtilis have functional ingredients that improve nutrition and health [B. subtilis, which provide an excellent source of data related to valuable favor/nutrients compounds [B. subtilis.industry . Some fod health , 8, 23. ompounds , 10. NevB. subtilis BJ3-2, finding that BJ3-2 cultured at different temperatures had various phenotypes on color  changed in the BT45 and CT53 compared to AT37  were significantly changed in the CT53 compared to BT45 , 356 DEPs (132 down-regulated and 224 up-regulated), and DEPs (224 down-regulated and 125 up-regulated) were identified in BP45 vs. AP37, CP53 vs. AP37, and CP53 vs. BP45, respectively (Fig. 4+ and pyruvic acid in B. subtilis. It has been shown that 64 metabolites were markedly up-regulated, and 68 were down-regulated in B45 compared to those in A37 (Fig. ed foods . Numerouompounds , 7–9, 11A37 Fig. . NotablyB. subtilis at different temperatures. Transcriptome analysis revealed more than 841 DEGs, 1970 DEGs, and 1043 DEGs that participated in soy sauce-like and soybean flavor accumulation in BT45 vs. AT37, CT53 vs. AT37, and CT53 vs. BT45, respectively. Moreover, the significantly changed metabolites involved in sulfur metabolism, cysteine and methionine metabolism, and pyrimidine metabolism in the strains cultured at 45℃ and 53℃ may cause soy sauce-like and soybean flavor accumulation in B. subtilis BJ3-2. These results further our understanding of the mechanism of soy sauce-like flavor and soybean flavor in B. subtilis. They also provide a series of candidate genes and metabolic engineering to enhance the production of flavor compounds.In summary, we combined transcriptomics, proteomics, and metabolomics analyses to explore the mechanisms underlying the flavor accumulation in Additional file 1: Fig. S1. Growth and physical characteristics. A: The color of fermentation broth; B: The OD value of fermentation broth; C: Gram staining of BJ3-2 cultured at 37°C; D: Gram staining of BJ3-2 cultured at 45°C; E: Gram staining of BJ3-2 cultured at 53°C. Fig. S2. Expression density distribution. Fig. S3. Data quality control of RNA-seq. A:Pearson correlation between smaples; B: Principal component analysis. Fig. S4. Analysis of DEGs. A: Volcano plot for group AT37 vs BT45; B: Volcano plot for group AT37 vs CT53; C:Volcano plot for group BT45 vs CT53; D: Cluster analysis of DEGs using H-cluster method. Fig. S5. Electrophoretograms of protein expressions. Fig. S6. Heat map analysis of DPGs in the AP37 vs BP45 A, AP37 vs CP53 B and BP45vs CP53 C. Fig. S6. Heat map analysis of DPGs in the AP37 vs BP45 A, AP37 vs CP53 B and BP45vs CP53 C. Fig. S7. Volcano plot analysis of DEPs in the AP37 vs BP45 A, AP37 vs CP53 B and BP45vs CP53C. Fig. S8.KEGG enrichment analysisfor group A37 vs B45 A, A37 vs C53 B and B45 vs C53 C.Additional file 2: Table S1. The data set summary of transcriptome sequence.Additional file 3: Table S2. The summary of DEGs.Additional file 4: Table S3. GO enrich for BT45vsAT37.  Additional file 5: Table S4. GO enrich for CT53vsAT37.Additional file 6: Table S5. GO enrich for CT53vsBT45Additional file 7: Table S6. KEGG enrich for BT45vsAT37.Additional file 8: Table S7. KEGG enrich for CT53vsAT37.Additional file 9: Table S8. KEGG enrich for CT53vsBT45.Additional file 10: Table S9. List of DEPs.  Additional file 11: Table S10. GO annotation enrichment analysis in BP45_vs_AP37. Additional file 12: Table S11. GO Annotation Enrichment Analysis in CP53_vs_AP37Additional file 13: Table S12. GO Annotation Enrichment Analysis in CP53_vs_BP45.Additional file 14: Table S13. Differentially expressed metabolites.Additional file 15: Table S14. Information of correlation analysis by multiple omics.


The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. Since a large portion of the world’s population is currently unvaccinated or incompletely vaccinated and has limited access to approved treatments against COVID-19, there is an urgent need to continue research on treatment options, especially those at low cost and which are immediately available to patients, particularly in low- and middle-income countries. Prior in vitro and observational studies have shown that fluoxetine, possibly through its inhibitory effect on the acid sphingomyelinase/ceramide system, could be a promising antiviral and anti-inflammatory treatment against COVID-19. In this report, we evaluated the potential antiviral and anti-inflammatory activities of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and against variants of concern in vitro, i.e., SARS-CoV-2 ancestral strain, Alpha B.1.1.7, Gamma P1, Delta B1.617 and Omicron BA.5. Fluoxetine, administrated after SARS-CoV-2 infection, significantly reduced lung tissue viral titres and expression of several inflammatory markers . It also inhibited the replication of all variants of concern in vitro. A modulation of the ceramide system in the lung tissues, as reflected by the increase in the ratio HexCer 16:0/Cer 16:0 in fluoxetine-treated mice, may contribute to explain these effects. Our findings demonstrate the antiviral and anti-inflammatory properties of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and its in vitro antiviral activity against variants of concern, establishing fluoxetine as a very promising candidate for the prevention and treatment of SARS-CoV-2 infection and disease pathogenesis. However, the use of these treatments may be held back due to either their medical indications , contraindications  antidepressants, particularly fluoxetine and fluvoxamine, could be beneficial against COVID-19 ,10,11, aAlthough the mechanisms underlying this potential therapeutic effect of fluoxetine and fluvoxamine against COVID-19 are likely to be multiple and interrelated, several studies support that the interaction with the acid sphingomyelinase (ASM)/ceramide system, resulting in both antiviral and anti-inflammatory effects, may be a prominent one . First, Among SSRIs, the magnitude of the in vitro inhibition of ASM appears to correlate with the magnitude of the in vitro antiviral effect against SARS-CoV-2 ,15,40  promoter (the K18-hACE2 mouse) was generated to study SARS-CoV pathogenesis . Recent In this report, we tested the potential antiviral and anti-inflammatory activities of fluoxetine against SARS-CoV-2 in a K18-hACE2 mouse model of infection, and against several variants of concern in vitro, and tested the hypothesis of the implication of ceramides and/or their derivatives hexosylceramides.10 TCID50/mL 6.19 (±0.14) vs. 4.04 (±0.44); Mann-Whitney test p = 0.0012) and day 6 (log10 TCID50/mL 4.22 log10 (±0.19) vs. 3.28 (±0.17); p = 0.0048). Moreover, viral RNA in lung homogenates was significantly reduced at day 2 post-infection in fluoxetine-treated mice compared to vehicle-treated mice (9.91 × 104 (± 4.15 × 104) vs. 1.04 × 106 (± 6.60 × 105); p = 0.0310) (4 (± 2.50 × 104) vs. 3.85 × 105 (± 1.22 × 105); p = 0.0295)  C. Viral  0.0295) . These r−4 (± 4.09 × 10−4) vs. IL-6 1.06 × 10−1 (± 4.88 × 10−2); p = 0.0017) and day 6 post-infection (IL-6 3.01 × 10−3 (± 1.58 × 10−3) vs. IL-6 2.31 × 10−3 (±6.11 × 10−3); p = 0.0140), and other pro-inflammatory cytokines at day 2 post-infection, including TNFα (8.07 × 10−4 (± 1.21 × 10−4) vs. 2.24 × 10−3 (± 3.70 × 10−4); p = 0.0004), CXCL10 (2.44 × 10−3 (± 7.17 × 10−4) vs. 9.99 × 10−2 (± 4.14 × 10−2); p < 0.0001) and CCL2 (8.83 × 10−4 (± 1.21 × 10−4) vs. 2.24 × 10−3 (± 3.70 × 10−4); p < 0.0001). A reduction in pro-inflammatory cytokine expression did not appear to be solely related to reduced viral load in fluoxetine-treated mice. Indeed, the difference in levels of IL-6 and TNFα between fluoxetine-treated and vehicle-treated mice was still significant after adjusting successively for viral load and viral RNA at both day 2 and day 6  and vehicle-treated mice  (As shown in nd day 6 . In addi 0.4508) . These r 0.4508) . To eval 0.4508) , indicatn = 4)) as a sign of critical illness [p = 0.0052)  .p = 0.0186) and the ratio HexCer 16:0/Cer 16:0 (0.40 (±0.05) vs. 0.28 (±0.03); p = 0.0408). There were no significant between-group differences in ceramides 16:0 and sphingomyelin 16:0 levels  and human lung epithelial cells ,17,18. TBy showing a significant increase in lung levels of hexosylceramides 16:0 and in the ratio HexCer 16:0/Cer 16:0 in fluoxetine versus vehicle-treated mice, our results suggest that the ceramide system might be implied in the observed antiviral and/or anti-inflammatory effects. Further analysis is now required to evaluate overtime, in comparison to baseline levels, those lipids dynamics during SARS-CoV-2 infection. Nevertheless, these findings are consistent with preclinical data indicating that the inhibition of the ASM/ceramide system by fluoxetine prevents infection of Vero E6 cells with pp-VSV-SARSCoV-2 spike  and that90 target, obtained from Calu-3 cells, in the lung tissues by day 1 at 40 mg/d and by day 3 at 20 mg/d. It is also supported by an experiment conducted in healthy volunteers showing that oral use of another FIASMA antidepressant, amitriptyline, whose blood half-life is about 20 h, prevents infection of freshly isolated nasal epithelial cells several hours after having taken the treatment, which is restored after the reconstitution of ceramides in these cells [Importantly, because the blood half-life of fluoxetine is about 2 to 4 days in humans and experimental data showed a human fluoxetine concentration lung:plasma split of 60:1 , fluoxetse cells .Fluoxetine is a prescription medication with a number of potential side effects and drug interactions, which are detailed in the Summary of Product Characteristic (SmPC)  and shouTo the best of our knowledge, this study is the first to identify fluoxetine as a potential anti-SARS-CoV-2 medication in an animal model, through mechanisms that result in both antiviral and anti-inflammatory properties. Since fluoxetine is easy to use as a once-a-day pill, shows high safety margins and good tolerability, is widely available at low cost, and has, among SSRIs, both the greatest in vitro inhibition effect on the ceramide system and antiviral effect ,15,34, tAll described experiments involving SARS-CoV-2 infections have been entirely performed and processed in a Biosafety Level 3 (BSL-3) facility. Prior samples from infected cells being taken out of the BSL-3 for subsequent analysis, and all SARS-CoV-2 inactivation procedures were validated and approved by Institute of Pharmacology and Structural Biology (IPBS) biosecurity committee.Virus productions were performed as previously described . Briefly2 atmosphere-enriched chamber. Serial 2.5-fold dilutions (from 10−1 to 10−6.5) of the samples were prepared in DMEM and used to infect Vero E6 cells; each dilution was tested in four replicates. The plates were incubated for at least 96 h and observed to monitor the development of cytopathic effect using an EVOS Floid microscope (Invitrogen). Viral titres, expressed as median tissue culture infectious dose (TCID50/mL), were calculated according to both Reed and Muench and Karber methods based on three or four replicates for dilution [The day prior to infection, 50,000 VeroE6 cells per well were seeded in 96-well tissue culture plates using 10% FBS DMEM, and then incubated overnight at 37 °C in a humidified, 5% COdilution .2O2 at 10 mM concentration and stored at −20 °C until use. The day prior to infection, 50,000 Vero E6 or A549-ACE2-TMPRSS2 cells were seeded in 96-well tissue culture plates in 10% FBS DMEM supplemented with 10 mM HEPES (Gibco™) and 1% penicillin-streptomycin (Gibco™), and then incubated overnight at 37 °C in a humidified, 5% CO2 atmosphere-enriched chamber. One day later, cells were treated with fluoxetine (12.5 µM) for 2 h prior infection, and then infected with SARS-CoV-2 strains at indicated MOI in 90 µL DMEM supplemented with 10 mM HEPES, 1% penicillin-streptomycin and 1% L-Glutamine for 1 h at 37 °C. Then, culture medium was completed with 90 µL of culture medium containing fluoxetine at 25 µM.For in vitro experiments, fluoxetine hydrochloride  was dissolved in H® permeable supports  and cultured with organoid medium [5 PFU/ALI) on the apical side for 4 h, washed with 150 µL of warmed PBS and then incubated for 72 h while maintaining fluoxetine on the basolateral side. Apical side of each ALI was washed with 150 µL of warm PBS to assess live viral load by TCID50 assay on Vero E6 cells. ALI were fixed with 4% paraformaldehyde overnight before being taken out of the BSL-3. For staining, cells were permeabilized with 0.2% Triton X-100, then membranes were excised in 2 mL Eppendorf tubes containing blocking buffer (PBS 3% BSA) and incubated for 20 min at room temperature. Primary antibodies were incubated overnight at 4 °C  and Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany (License 81-02.04.2021.A112) and performed according to the European Union animal protection directive (Directive 2010/63/EU).C57BL/6 male mice  received 5 mg/kg fluoxetine  orally and serum levels of fluoxetine and its active metabolite desmethylfluoxetine were measured over time as indicated . To deteMouse infection studies were approved by local and national ethic committees  and performed according to local guidelines  and the European Union animal protection directive (Directive 2010/63/EU).5 PFU of SARS-CoV-2 (strain BetaCoV/France/IDF0372/2020). Thereafter, 4 h post-infection and then once daily, the first group received the vehicle  treatment and the second group received fluoxetine (40 mg/kg i.p.)  to 16 (day 2) per group and time point) were infected under a short anaesthesia (isoflurane 4%), by the intranasal route with 10kg i.p.) . During 7 to 100 copies) and qPCRs were performed in TaqMan Universal PCR Master Mix (Thermo Fisher Scientific). qPCRs for gene expression and viral load were performed in triplicate and assessed with an ABI 7500 real-time PCR system . Primers and probe sequences are provided in For each mouse, 150 µL of lung tissue homogenate was mixed with 1 mL TRIzol Reagent (Invitrogen) and stored at −80 °C at least 48 h before being taken out the BSL-3 facility. Total RNA was extracted, reverse-transcribed and gene expression levels of cytokines and chemokines were assessed as previously described . Viral lv:v) containing the internal standards 17:0 ceramide (d18:1/17:0), d31-16:0 sphingomyelin (d18:1/16:0-d31) and 17:0 glucosyl(β) ceramide (d18:1/17:0)  were added. Extraction was facilitated by incubation at 48 °C with gentle shaking (120 rpm) overnight. To reduce interference from glycerolipids, samples were saponified with 150 μL 1 M methanolic KOH for 2 h at 37 °C with gentle shaking (120 rpm) followed by neutralization with 12 μL glacial acetic acid. After centrifugation at 2200 g for 10 min at 4 °C, organic supernatants were evaporated to dryness using a Savant SpeedVac concentrator . Dried residues were reconstituted in 200 μL acetonitrile/methanol/water (47.5:47.5:5 (v:v:v), 0.1% formic acid) and subjected to LC-MS/MS sphingolipid quantification applying the multiple reaction monitoring (MRM) approach. Chromatographic separation was achieved on a 1290 Infinity II HPLC  equipped with a Poroshell 120 EC-C8 column  guarded by a pre-column  of identical material. MS/MS analyses were carried out using a 6495 triple-quadrupole mass spectrometer (Agilent Technologies) operating in the positive electrospray ionization mode (ESI+). Chromatographic conditions and settings of the ESI source and MS/MS detector have been published elsewhere [m/z 520.5 → 264.3), SM 16:0 (m/z 703.6 → 184.1), and HexCer 16:0 (m/z 700.6 → 264.3) subspecies, as determined with MassHunter software , were normalized to those of their internal standards. Quantification was performed with MassHunter Software . Further, the determined Cer, SM and HexCer amounts were normalized to the actual protein content of the tissue homogenate used for extraction determined by the Coomassie (Bradford) Protein Assay .Sphingolipids were extracted from lung tissue homogenates (20 µL) as previously described . To thislsewhere . Peak ar4 cells per well and incubated at 37 °C. The following day, cells were pre-incubated with or without fluoxetine (12.5–25 µM) for 1 h before treatment with various activators of the NF-kB and/or IRF pathways including TNF-α (10–0.1 ng/mL), IFN-α (10–0.1 ng/mL), and poly I::C . After 24 h of incubation, supernatant was collected and the presence of SEAP and Lucia reporting respectively for NF-kB and IRF activation was quantified following incubation with SEAP and Lucia enzyme substrates Quanti-blue and Quanti-luc, respectively, according to the manufacturer’s instructions .To investigate whether fluoxetine modulates NF-kB and interferon pathways, we used A549–Dual cells carrying both an NF-κB-driven secreted phosphatase alkaline (SEAP) gene and an IRF-driven Luciferase reporter genes . Cells were plated in 96-well plates at 5 × 10t-tests or Mann-Whitney tests when assumptions were not met. For all associations, we performed residual analyses to assess the fit of the data, checked assumptions, and examined the potential influence of outliers. Outliers were defined as having values outside the 1.5 interquartile range (IQR). Analyses were carried out with GraphPad Prism 9 and R Software, version 4.1.3. Statistical significance was considered when two-sided p < 0.05.All data in the text and figures are expressed as mean (±SEM). All means were compared using


Pseudomonas aeruginosa, a novel catechol–zingerone conjugate (1) linked via a non-hydrolyzable 1,2,3-triazole linker was synthesized and subjected to biological evaluation based on the Trojan horse strategy. To enhance the efficacy, catechol, a xenosiderophore, utilized by P. aeruginosa for iron assimilation, and the dietary phytochemical zingerone, known for its anti-virulent activity against Pseudomonas aeruginosa, were exploited in the present study. Theoretical validation of conjugate (1) was conducted by in silico molecular docking analysis to determine the interaction with outer membrane transport receptor PirA and quorum sensing signal receptors. In addition, nine-fold binding affinity of Conjugate (1) toward PirA (5FP2) in comparison to its natural ligand catechol with D-score −1.13 Å authenticated the designed Trojan horse drug. Conjugate (1) showed stronger anti-virulent activity than zingerone; hence, it exhibited a promising anti-biofilm efficacy as assessed by crystal violet assay and visualized by FESEM toward P. aeruginosa. Encouraging results against P. aeruginosa in terms of quorum sensing regulated virulence factors, motility phenotypes, and biofilm formation with no cell cytotoxicity and could help open hitherto unexplored possibilities of establishing Trojan horse drugs as a successful approach against multidrug resistance in P. aeruginosa.To address the issue of multidrug resistance in  Pseudomonas aeruginosa, one of the Gram-negative ESKAPE pathogen, is responsible for 10% of the hospital-acquired infections, including chronic cystic fibrosis , the second-most common siderophore of P. aeruginosa, is reported to be conserved and produced by all the strains of P. aeruginosa and Burkholderia cepacia. Modified pyochelin was utilized for the construction of pyochelin–fluoroquinolone conjugates bearing a cleavable linker , having less chances of steric hindrance during conjugation.There are several inherent problems which need to be addressed before the development of a hybrid drug especially if the agent is directed at Gram-negative pathogens. Limited cellular penetration across the dual layer of Gram-negative bacteria is the first major concern for hybrid agents that have molecular mass of > 600 g/mol. Using catechol, a siderophore entity, as a starting point for drug conjugate may enable solving the problem of penetration and may also enhance the permeability by exploiting the iron uptake system. Studies have reported that siderophore components in conjugates do not necessarily have to identically replicate the natural siderophores . Also, PThe other problem lies within the fundamental idea of covalently linking two entities together. The point of attachment and physicochemical properties of the chosen linker are crucial for the overall activity of the hybrid. The linker group connects the vector with the drug and hence acts as a key component in the function and effectiveness of the conjugate. Linkers can be cleavable either by enzyme catalysis or by hydrolysis and non-cleavable which are stable to metabolic degradation. The prerequisite attributes of an ideal linker includes less toxicity, high stability in the biological system, and specific release of the drug. Triazole, being stable to metabolic degradation along with its improved solubility due to formation of hydrogen bonding, can be used as a non-cleavable linker.P. aeruginosa highly susceptible for killing with antibiotics and by serum and phagocytes, components of innate immunity  is mainly found in dry ginger root and also produced by gingerols and shogoals. The retro-aldol reaction results in the conversion of gingerol (component of ginger) into zingerone on cooking. It has been observed in our previous research that zingerone attenuates cell surface properties, making immunity . Studiesimmunity . TherefoP. aeruginosa. Past studies have demonstrated the presence of catecholate-binding outer membrane proteins  and the quorum sensing signal receptor proteins  in P. aeruginosa  of antibiotics, hence overcoming the menace of development of antibiotic resistance.With the aim of developing more potent and better targeted drugs by following the Trojan horse strategy, the present study is designed to synthesize a hybrid drug, catechol–zingerone, to be used against ruginosa . In ordehttp://www.rcsb.org. For RhlR, the targeted amino acid sequence P54292 was obtained from the UniProt KB database (http://www.uniprot.org/) to perform homology modeling.The 3D crystallographic structure for Pir A (PDB: 5FP2), LasR (PDB: 2UV0), PqsR (4JVI), TraR (1HOM), and RhlR HM (P54292) was retrieved from a protein data bank To prepare the target protein, the protein structure was analyzed, reprocessed, and refined using the protein preparation protocol. The protocol was performed by removal of water molecules and protonation of titratable residues to stabilize the receptor protein. Furthermore, protein structure integrity was assessed and checked for its missing residues, followed by insertion of loop regions and missing atoms near the active site, using a loop builder tool. Furthermore, co-factors and external ligands present in the crystal structure were deleted and extracted out. Ultimately, final protein was saved in .mol format as a receptor. At the same time, homology modeling was performed for the RhlR selecting a suitable template for each target sequence using the BLAST tool.1) and natural ligands . However, energy calculation and optimization of structural geometry were performed using VLife MDS 4.6 software. The two-dimensional (2D) structures were transformed into three-dimensional (3D) structures by using the converter module of the VLife module. The 3D structures were then subjected to energy minimization and geometry optimization using the Merck Molecular Force Field (MMFF) method. Conformers with lowest energy were selected for docking simulation studies. The comprehensive and integrated graphical user interface program of the Vlife MDS 4.6, that is, “Bio Predicta module” was used to prepare, run, and analyze the docking simulations on the HP Pentium IV 2.80 GHz Processor/Microsoft Win XP Home Edition system.One of the important determinants for a successful docking is the structure of the ligand. The ChemDraw ultra 12.0 software was used to draw the structure of catechol–zingerone conjugate (1H NMR spectra were recorded on a Bruker AV-400 spectrophotometer at 400 MHz or a Bruker AV-500 spectrophotometer at 500 MHz. 13C NMR spectra were recorded on a Bruker AV-500 spectrophotometer at 125 MHz. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts were reported in parts per million (ppm) relative to an internal standard (tetramethylsilane). Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (b). Mass spectra were recorded on an LCMS-Q-TOF (Waters) by means of the ESI method. The elemental analysis for C, H, and N was carried out with an elemental analyzer GmbH VarioEL. Purity of the compound was determined by reverse-phase high-performance liquid chromatography  to be >95%. A flow rate of 1.0 ml/min was used with the mobile phase of acetonitrile in water with 0.1% modifier (orthophosphoric acid. v/v).All reagents and solvents were purchased from Merck and Aldrich companies without any purification. The reaction progress and the purity of synthesized compounds were monitored by thin-layer chromatography (TLC) using silica gel plates with Fluorescence F254 and UV visualization. Flash chromatography was performed by using a 230–400 mesh silica gel and the indicated solvent system. 3 (240 mg) in anhydrous CH2Cl2 (5 ml) at 20°C was dropwise added a solution of pentafluorophenyl ester 2 (1.0 g) in a minimum volume of anhydrous CH2Cl2 (5 ml). The resulting mixture was stirred at 20°C for 3 h and TLC-monitored. On completion of the reaction, the solvent was evaporated under reduced pressure. The crude mixture was purified on a silica gel column (hexane: ethyl acetate 8:2) to afford compound 4 in the form of yellow oil in 80% yield (m.p.: 148°C).To a stirred solution of 1-azido propylamine 1H NMR : 7.53–7.58 , 7.40–7.43 , 7.31 ,7.01 , 6.94 , 3.60, 3.44 , and 1.92 .13C NMR : δ 162.88, 146.75, 144.26, 139.18, 129.39, 128.43, 125.78, 121.81, 121.42, 117.02, 116.83, 111.13, 48.64, 36.67, and 28.32.23H20N4O3: calcd. 400.44, found 400.55.MS (ESI): m/z 400.15 [M + H+] HRMS (ESI): C−1): 3,426, 2,921, 2,100, 1,648, 1,593, 1,536, 1,501, 1,456, 1,250, 1,210, 1,052, 1,019, 747, and 700.IR  and propargyl bromide  were added dropwise. The mixture was stirred at 60°C for 24 h and quenched with H2O at 0°C. Acetone was removed under reduced pressure, and the product was extracted with CH2Cl2. The organic layer was washed with saturated NaHCO3 and brine, dried over MgSO4, filtered, and concentrated. The product was obtained as brown solid (1.1 g) in quantitative yield (1.1 g). (m.p.: 197°C).To a stirred solution of zingerone  in dry acetone (20 ml), K1H NMR : δ 6.94, 6.73 , 6.71 , 4.72 , 3.86 , 2.84, 2.74 , 2.49 , and 2.13 .13C NMR : δ 207.64, 149.05, 144.53, 134.85, 119.62, 114.39, 112.40, 79.43, 77.92, 56.07, 55.33, 44.23, 29.61, and 28.63.13H14O3: calcd. 218.25, found 218.MS (ESI): m/z 218.25 [M + H+] HRMS (ESI): C−1): 3,434, 3,248, 2,915, 2,125, 1,706, 1,589, 1,513, 1,450, 1,423, 1,370, 1,329, 1,249, 1,160, 1,134, 1,021, 802, 715, 621, 543, and 447.IR  and alkyne 6  in THF (1 ml per 0.1 mmol) was added successfully 0.8 M aqueous solution of copper sulfate (1 eq.) and sodium ascorbate (4.7 eq.). The suspension was sonicated and stirred under argon at room temperature (21–23°C). The reaction mixture was filtered on a celite pad and purified on a silica gel column (CH2Cl2: MeOH) to afford the expected conjugate. For the release of the catechol moiety, conjugates were dissolved in methanolic HCl (6 ml) and further stirred overnight before being evaporated under reduced pressure. The residue was suspended in a minimum of THF, and cyclohexane was added until precipitation of expected conjugate (103 mg) in 60% yield. (m.p.: 248°C).To a solution of azido-catechol 1H NMR : δ 12.68 , 9.13 , 8.83 , 8.23 , 7.27 ,6.99 , 6.92 , 6.83 , 6.68–6.71 , 5.05 , 4.44 , 3.73 , 3.30–3.33 , 2.69–2.75 , 2.10–2.15 , and 2.09 .13C NMR : δ 207.67, 169.86, 149.53, 148.91, 146.11, 145.54, 142.73, 134.27, 124.50, 119.75, 118.72, 117.79, 117.05, 114.82, 113.89, 112.35, 61.84, 55.29, 47.23, 44.28, 36.25, 30.56, 29.62, and 28.65.24H28N4O6: calcd. 468.20, found 469.3.MS (ESI): m/z 468.20 [M + H+]. HRMS (ESI): C−1); 3,386, 2,928, 1,710, 1,641, 1,594, 1,545, 1,513, 1,459, 1,328, 1,262, 1,228, 1,158, 1,139, 1,010, 1,031, 835, 802, 742, 630, 591, and 546.IR , 12.53 min, 95.5% purity.HPLC analysis: ACN:HP. aeruginosa PAO1(MTCC-3541) was obtained from Dr. Barbara H. Iglweski, Department of Microbiology and Immunology, University of Rochester, New York (United States). Agrobacterium tumefaciens A136 was obtained from J. Handlesman, Wisconsin University  and was used as a biosensor strain. All the strains were maintained in glycerol (50%) and stored at −80°C. Fresh stocks were subcultured for every new experiment. Working solutions of compounds were prepared in 5% (v/v) DMSO supplemented with 0.01% Tween 80.A standard strain of 1, Compound 6, zingerone 5, and 10% DMSO was added into the wells. Color change was observed after 12 h at room temperature.To check the iron chelation activity of the synthetic catechol–zingerone conjugate, the CAS agar plates were prepared . Wells  was used, whereby the diameters of zones of inhibition around the wells with the test compounds on the surface of P. aeruginosa were measured.To assess the antimicrobial activity against Pseudomonas aeruginosa PAO1 was grown overnight in Luria Broth (14-16h) and was centrifuged at 4°C for 10° min at 3,000 ×g to obtain the cell-free supernatant. AHLs were extracted from the supernatant using an equal volume of acidified ethyl acetate (0.01% acetic acid) and was kept overnight at 4°C. An organic layer was separated and concentrated under reduced pressure. The QS inhibition effect (QSI) was determined by performing the qualitative assay using A. tumefaciens A136  was added to 200 µl of the cell-free supernatant. After incubation, an equal volume of trichloroacetic acid (TCA) was added to stop the reaction. Absorbance of the supernatant was measured after centrifugation at 420 nm.Proteolytic activity was quantified in the cell-free supernatant . In brieThe elastolytic activity was measured using elastin Congo red as the substrate . BrieflyAlginate concentration was assessed in the supernatant . BrieflyPyochelin was estimated by using an Arnow assay . A volum8 CFU/ml) were used (To quantify the concentration of cell-free hemolysin (CFH) and cell bound-hemolysin (CBH), the cell-free supernatant and cells (10ere used . Briefly8 cfu/ml) grown in the presence and absence of conjugate 1, compound 6, and zingerone alone were harvested, washed, and resuspended in PBS (Ph-7.2). Initial OD (i) was measured at 600 nm. A volume of 0.35 ml of xylene was added, and cells were vortexed for 1 min. After phase separation, final O.D of the aqueous phase was measured at 600 nm and expressed as O.D. (f) Surface hydrophobicity of each strain was expressed as percent hydrophobicity.The method of 1, compound 6, and zingerone 5 at a concentration of 50 µM on motility phenotypes, the cells of P. aeruginosa grown in presence and absence of sub MIC of treatment regime were point-inoculated on swarm agar plates . Swarming motility was determined by measuring turbid circular zones after 24 h of incubation at 37°C.To study the effect of conjugate P. aeruginosa were inoculated on swim agar plates . After 24 h of incubation at 30°C, the plates were observed for the swimming motility around the point inoculation.To assess the swimming motility, treated and untreated cells of P. aeruginosa cells with and without treatment were stabbed in LB plates (bacto agar 1%). After 24 h of incubation at 37°C, a hazy zone of growth at the interface of the agar was measured.To assay twitching motility, 6 CFU/ml) of P. aeruginosa. Conjugate 1, compound 6, and zingerone alone were added to tubes at sub-MIC concentration (50 µM). A tube without conjugate 1 served as the control. The tubes were incubated for 24 h. The media containing loose planktonic cells were discarded and washed thrice with phosphate-buffered saline (PBS). After washing, the adherent cells were stained with 0.1% crystal violet (CV). After 30° min, the tubes were washed with distilled water to remove excess stain. The color intensity was examined to assess biofilm formation. Adherence ranged from weak (+) to strong (+++).A volume of 3 ml LB in polypropylene microcentrifuge tubes  was inoculated with 50 µl of an overnight grown culture . Diluted mid-log phase cells of P. aeruginosa (1 × 106 CFU/ml) were added to different wells of the microtiter plate and supplemented with sub-MIC concentration, that is, 50°µM of conjugate 1, compound 6, and zingerone 5 alone. Bacterial culture without treatment served as the control. The attached biofilm cells were stained with CV after discarding media containing planktonic cells alone with the PBS washings. Absorbance was measured on each day after elution of stain with ethanol. The wells were processed in a similar way up to 7°days.Biofilm inhibition ability of conjugate 6 CFU/ml) was added to wells of the microtiter plate. The preformed peak (4°days old) biofilm was treated with 50 µM of conjugate 1, compound 6, and zingerone alone. Untreated wells served as the control. The media containing loose planktonic cells were discarded and washed thrice with phosphate-buffered saline (PBS) followed by staining with CV for 30 min. Stain was eluted with ethanol followed by absorbance at 545 nm.A volume of 100 μl of diluted culture  was added to each well and incubated in dark for 30° min. Excess dye was removed with distilled water, and the stain was eluted with DMSO. Absorbance was measured at 490 nm in an ELISA reader (REMI).The interference of conjugate ng assay . The peaP. aeruginosa were formed on sterile conditions on catheter (1 × 1°cm). The biofilms were grown in the absence and presence of conjugate 1, compound 6, and zingerone. The peak day biofilms were fixed with 2.5% (v/v) glutaraldehyde in PBS for 1 h at room temperature in dark. After washing, dehydration was performed in ethanol with different concentrations . Catheters were dried, gold-coated, and viewed under a field emission scanning electron microscope .Microscopic analysis of biofilms was performed by SEM . Biofilm1, compound 6, and zingerone on membrane permeability was detected by using a crystal violet uptake assay (P. aeruginosa (1 × 106 CFU/ml) were prepared in LB broth. The cells were harvested, washed twice, and suspended in 50 mM PBS (pH 7.4). Conjugate 1 at the concentration of 100 µM was added to the cell suspension and incubated at 37°C for 30 min. Suspension without treatment served as the control. The cells were harvested at 10,000 rpm for 5 min and suspended in PBS containing 10 μg/ml of crystal violet. Aliquots of 1 ml were removed at different time intervals  and centrifuged at 10,000 rpm for 5°min, after which OD590 of the supernatant was measured. The OD value of crystal violet solution, which was originally used in the assay, was considered 100% excluded. The percentage of crystal violet uptake was calculated using the following formula:The influence of conjugate ke assay . SuspensP. aeruginosa was determined by using the Bradford reagent 6 CFU/ml) were harvested at 8,000 rpm, washed twice with NaCl (0.85%), and finally suspended in 1 ml of saline in the presence of treatment regimes and incubated at 37°C. Suspension without treatment served as the control. After the incubation of 60 and 120°min, aliquots of 1 ml were removed and centrifuged at 10,000 rpm for 5 min. Absorbance of the supernatant was then measured at 260 nm. The results were expressed in the form of OD recorded at each time interval. To confirm membrane disintegration of P. aeruginosa by the conjugate, released membrane proteins were also detected by SDS-PAGE. Intensities of separated bands increased from 60 to 120 min and showed a distinct banding pattern as compared to the control (untreated).Enumeration of release of cellular materials from ad libitum. The ethical clearance to conduct the experiment was approved by the Institutional Animal Ethics Committee of Panjab University, Chandigarh, India . All the experimental protocols were followed according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.Female 6–8°week-old BALB/c mice (weighing 20–30 g), bred and procured from the Central Animal House, Panjab University, Chandigarh, India, were used in the present study. The mice were housed in polypropylene cages and bedded with dry and clean husk with proper ventilation of the room. The animals were fed on standard pellets of antibiotic-free synthetic diet  and water 1, compound 6, and zingerone alone were evaluated for cytotoxicity using the 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay method of 5 cells/ml) in a 96-well plate and cultured overnight to obtain a monolayer. The cells were then treated with conjugate 1, compound 6, and zingerone alone at sub-MIC concentration and were incubated at 37°C for 24 h in a CO2 incubator. After incubation, the cells were exposed to MTT solution (5 mg/ml) for 4 h at 37°C. Media were removed, and the formed formazan crystal was solubilized with 200 μl of 100% ethanol. Absorbance at 490 nm was measured. Percent viability of cells was calculated by comparison to that of untreated control cells.The conjugate To evaluate the hemocompatibility of the conjugate, 1% RBC suspension was used. 100°μl of the blood suspension was mixed with 100 μl of the conjugate in a U- or V-shaped microtiter plate. After 24 h of incubation, the settling patterns of single and agglutinated red blood cells were observed with the help of a hand lens.8 cells/ml), 500 μl of pre-warmed serum was added and incubated at 37°C. The samples were taken out at 0, 1, 2, and 3 °h post incubation. Different dilutions were prepared and plated on MacConkey’s agar plates and incubated at 37°C to obtain the counts. The results were expressed as follows:(1) Completely serum-resistant: Organism surviving for 3 h in serum without showing any increase or decrease in viable count after 1 h.(2) Delayed serum-sensitive: Organism showing significant decrease in cell count after surviving for 1 h.(3) Promptly sensitive: No viable cells detected after 1 h.To 250 μl of treated/non-treated PAO1 cells  test using GraphPad Prism version 5.0. The p-value <0.05 was considered statistically significant .All the experiments were conducted in triplicate, and results were expressed as means ± standard deviation of different measurements. Significance of data was evaluated with one-way1) and catechol itself as the natural reference ligand. The ligand poses, docking scores, and involvement of active amino acid residues along with their bond distance for each and respective complexes are manifested in 1) with 5FP2 receptor.To clear our perplexities of using catechol as a carrier for zingerone penetration through the outer membrane, a molecular docking study was performed using surface receptor 5FP2) with a novel catechol–zingerone conjugate ( with a n1) and catechol, showed binding affinity toward the receptor indicated by D-score −10.78 and −1.13 Å, respectively. Conjugate (1) showed a nine-fold binding affinity toward 5FP2 in comparison to its natural ligand (catechol 1), with D-score −1.13 Å. This can be attributed to the stearic factor that is helping the conjugate to extend deep into the binding cavity via the formation of hydrogen bonding with amino acids Ser 101A and Arg 309A at a bond distance of 2.501 and 2.061 Å, respectively. Furthermore, conjugate (1) was found to be engaged with hydrophobic interactions with Leu 92A (2.534 Å), Pro 99A (3.030 Å), Ser 351A (3.877 Å), and Glu 353A (3.393 Å) and van der Waals forces with amino acids Glu 371A, Arg 309A, and Pro 99A by affording bond distance of 2.740, 3.001, and 2.901 Å  that regulate phenotypic expression of virulence factors and biofilm formation. Further research findings have clearly indicated the role of LasR (PDB: 2UV0), PqsR (4JVI), TraR (1HOM), and the RhlR HM (P54292) system in quorum sensing. Herein, efforts have also been made to understand the binding potential and molecular insights of hypothetically designed catechol–zingerone conjugate toward the enlisted proteins, before carrying out its synthesis. Natural inhibitors  of all the enlisted proteins from our previously published study were also taken as a reference molecule .1) toward all the receptors by affording D-score ranging from −20.62 Å to −78.59 Å. Docking studies revealed (1) was found to interact in a better way with 2UV0  and 1HOM  in comparison to reference molecules (3-oxo-C12-HSL and 3-oxo-C8-HSL). This can be associated with well accommodation of the conjugate (1) inside the binding cavity of 2UV0 and 1HOM and highlighting the importance of conjugation and steric factor in quorum quenching potential.Good binding affinity has been indicated by conjugate  from Detailed analysis of the best complex  strong anti-virulent property against Gram-negative organisms due to hydrophobic nature, 2) possess periplasmic target in Gram-negative bacteria, 3) biddable to synthetic modifications without affecting anti-virulent properties, and 4) decreased selection pressure responsible for resistance. Indeed, many examples of pyochelin–antibiotic and pyoverdin–antibiotic conjugates with several types of linkers are reported in the literature . However1) has been performed with complete characterization by 1H NMR, 13C NMR, and mass spectral analysis. The pentafluorophenyl ester (2) was synthesized according to previously published protocol in good overall yield of 80% (2) and azido propylamine (3) (4) in 80% yield with a molecular mass of 400.13 as identified by mass spectral analysis, and a characteristic IR peak at 2,100 cm−1 confirms the azide group as shown in 6) was synthesized by nucleophilic substitution reaction of propargyl bromide on zingerone in the presence of potassium carbonate as a base and was formed in quantitative yield showing the characteristic peaks at δ4.7 and δ2.4 in 1H NMR and C-C alkyne stretch at 2,125.10 cm−1 in FT-IR spectral analysis (4) was then coupled with the acetylene derivative of zingerone (6) as shown in 1). The prominent peak observed at δ8.2 confirmed the triazole group in conjugate (1), which was further subjected to deprotection using methanolic solution of HCl undergoing cleavage of diphenyl dioxole groups, leading to the free functional hydroxyl groups. The final conjugate was purified by column chromatography using CH2Cl2: methanol (5%).The disappearance of peaks in the aromatic region in 1H NMR spectra confirmed the release of diphenyl groups (1) was further confirmed by mass spectral analysis (m/z 468.30), FT-IR spectral analysis (The stepwise synthesis of the catechol–zingerone conjugate (d of 80% . The synmine (3)  were reaanalysis . The azil groups . The finanalysis , and eleanalysis . Compounanalysis .1) and the parent drug zingerone and compound 6 was scrutinized on CAS agar plates. Orange halo around the well containing catechol–zingerone conjugate represents the positive reaction, indicating iron binding affinity of the conjugate (1) (1), a siderophore mimic, showed iron chelation activity similar to pyochelin, a natural siderophore of P. aeruginosa, despite structural modifications. These results are in consensus with the in silico results validating strong interactions of the conjugate with outer membrane receptors that is, PirA. Prior literature has also documented that the siderophore component in a conjugate does not necessarily have to be identically a replicate of the natural siderophore  . The reserophore .P. aeruginosa with both conjugate (1) and parent drug zingerone  with zone diameter of 15 mm (1) observed in the present study might be due to the increased stability as well as solubility of hydrophobic parent drug zingerone after conjugation with catechol moiety through triazole linker.To decrease the selection pressure of antibiotics on bacteria, disarming the pathogen of its virulence instead of killing seems a more promising approach. Since virulence of hibition . Maximumof 15 mm . As per of 15 mm , but solof 15 mm . TherefoP. aeruginosa, the trail of QS regulated extracellular virulence factors required for the succession of adhesion, colonization and invasion to host cell, needs to be halted (1) as well as parent drug, but maximum reduction was showed by siderophore mimic  (1) than in parent drug (5) (1) (P. aeruginosa are known to lyse the RBCs for acquiring iron affecting the host innate response (1) was evaluated on alginate production. In addition, alginate helps in attachment of P. aeruginosa on surfaces by enhancing cell surface hydrophobicity  might be due to the increased membrane permeability through the iron acquisition pathway, thereby clogging the QS network.For completely hijacking the e halted . Since tre mimic . Pyocyantructure . There wture (1) . Proteasdrug (5) . Reductidrug (5) , but no drug (5) . It is kdrug (5) . Upto 40 (5) (1) . Similar (5) (1) . Hemolysresponse . Since ahobicity . Highly surfaces . PromineP. aeruginosa acts as one of the vital virulence determinant due to its role in colonization to establish biofilms (1) along with the parent drug zingerone was evaluated on swim, swarm, and twitch plates in vitro. In the present study, swarming, swimming, and twitching motilities of P. aeruginosa in the presence of 50 μΜ catechol–zingerone conjugate (1) were reduced significantly as found with the parent drug, indicating non-interference of catechol moiety with antimotility activity of zingerone  as well as parent drug was evaluated qualitatively. In the presence of conjugate (1), P. aeruginosa showed weak adherence as compared to control which showed strong adherence to polypropylene tubes (P. aeruginosa was studied up to 7 days. Results inferred that biofilm formation was inhibited in the presence of conjugate (1) as well as parent drugs (1), which might be due to enhanced cell penetration of zingerone through extracellular polymeric substances and stability of zingerone when conjugated to catechol, a xenosiderophore. A significant reduction in peak-day biofilm was observed in all the regimes, but maximum reduction was observed in conjugate indicating anti-biofilm activity . EPS, being the vital functional component of P. aeruginosa biofilm matrix, was quantified in the presence and absence of conjugate as well as parent drugs. Treatment with conjugate significantly deteriorated the EPS production, resulting in devitalization of biofilm matrix , which is indicative of reduced biofilm as compared to control  might be attributed to its ability to penetrate or disrupt the bacterial plasma membrane. To understand the underlying mechanism, crystal violet uptake assay was performed. The results suggested that treatment with the 100 μΜ catechol–zingerone conjugate (1) enhanced membrane permeability of P. aeruginosa (P. aeruginosa with catechol–zingerone conjugate 1 led to the release of intracellular contents represented as separate bands on SDS-PAGE (1) in the binding pocket of Las and Rhl receptor, thereby seizing the pathogen of its virulence by downregulating lasI and rhlI genes as shown by in silico studies. These results were found to be in agreement with the action of thymol as stated in previous study, depicting increased membrane permeability and release of cellular contents in the presence of thymol (Mode of action of the catechol–zingerone conjugate (ruginosa . FurtherSDS-PAGE . This mif thymol . Howeverex vivo efficacy of catechol–zingerone conjugate (1) in terms of hemocompatibility, cell cytotoxicity, and serum susceptibility. For the contemplation of the conjugate as a new treatment regime, hemocompatibility testing was performed. Results reflected that incubation of RBCs with conjugate (1) in round-bottom 96-well plate resulted in button formation, indicating hemostability as compared to the control with cell lysis  can interact with membrane receptor PirA and the quorum sensing signal receptors with low binding affinity, indicating less or no steric hindrance of catechol moiety on drug components. This conjugate (1) was synthesized using a non-hydrolyzable linker which showed iron chelation activity. Furthermore, conjugate (1) can inhibit QS-activity, reduce virulence factor production, and disrupt biofilm formation, which is crucial for pathogenesis of P. aeruginosa. This study proves that conjugate (1) has the potential of modulating the surface properties of P. aeruginosa, leading to its enhanced serum susceptibility with no cell cytotoxicity. To the best of our knowledge, this is the first report to investigate the potential of catechol zingerone conjugate (1) in attenuation of virulence factors and biofilm formation in P. aeruginosa. However, more detailed in vivo studies are required to make use of conjugate (1) in clinical practice.This work illustrates that conjugation of natural bioactive, that is, zingerone with catechol, a xenosiderophore of


These results provide a compelling strategy for developing new small molecules capable of disrupting the TRIP8b–HCN interaction.Major depressive disorder is a critical public health problem with a lifetime prevalence of nearly 17% in the United States. One potential therapeutic target is the interaction between hyperpolarization-activated cyclic nucleotide–gated (HCN) channels and an auxiliary subunit of the channel named tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b). HCN channels regulate neuronal excitability in the mammalian hippocampus, and recent work has established that antagonizing HCN function rescues cognitive impairment caused by chronic stress. Here, we utilize a high-throughput virtual screen to find small molecules capable of disrupting the TRIP8b–HCN interaction. We found that the hit compound NUCC-0200590 disrupts the TRIP8b–HCN interaction  Most antidepressants focus on monoaminergic neurotransmitters, but these drugs take several weeks to achieve their therapeutic effect and fail to treat some of the most debilitating aspects of MDD, including cognitive impairment , 2. As sssengers . HCN1 anssengers , 11.Multiple lines of evidence suggest that limiting hippocampal HCN channel function may be a relevant therapeutic target for the treatment of MDD , 14, 15.To avoid inhibiting cardiac HCN channels, our group has focused on a subunit of HCN named tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b) , 22, 23.Although initial reports suggested a complete lack of TRIP8b in cardiac tissue , 26, a mTo validate the TRIP8b–HCN interaction as a therapeutic target, we recently employed a chemogenetic approach to disrupt the interaction in mice . Followih) with a reversal potential of approximately −25 mV  so that more hyperpolarized voltages are required to maximally open the channel , which is complexed with an HCN2 carboxy-terminus peptide, showed that the critical interactions between the peptide and TRIP8b included the E67, N99, R236, N205, R206, N213, and N240 residues of TRIP8b  charges. The Glide  implemented in the Sybyl interface of Tripos  to obtaiin silico screen were tested in biochemical assays to characterize biological activity. The molecular mechanism of HCN binding to TRIP8b has been elucidated by determination of the cocrystal structure of the carboxyl-terminal SNL sequence (SRLSSNL) of HCN2 bound to TRIP8b . The docked pose of NUCC-0200590 bound to TRIP8b is shown in Figure 1B.The 30 potential hits obtained from the aforementioned o TRIP8b . The como TRIP8b  and comp± 0.9 μM A. The doin vitro and in vivo studies methanamine using hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was not successful, so alternative approaches were explored. Though several early transition-metal complexes, especially from zirconium and titanium, have been used as catalysts in direct coupling of deactivated acids in high yields, the conversion was very low in our case. Conversion of the acid to an acid chloride before amidation was successful albeit with low yield. A larger set of coupling reagents were screened and O-(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate was identified as the best coupling reagent and produced the acetamide 6 in 66% yield. Suzuki coupling with (4-fluorophenyl)boronic acid afforded the final compound NUCC-0200590 in excellent yield. Conversion to the formic acid (FA) salt was achieved by exposure to the FA modifier in prep HPLC purification or to the hydrochloric acid (HCl) salt by treating the free base with 4M HCl in dioxane.We developed a synthetic route for compound NUCC-0200590 to prepare enough material needed for hit validation,  studies . Alkylat studies  with eth1H-NMR. On stirring the free amine in 4M HCl in dioxane, the compound was converted to the HCl salt as a white solid  NMR. To improve the aqueous solubility, we synthesized and characterized two salt forms of NUCC-0200590. The FA salt form was recovered after reverse-phase preparative HPLC while using 0.1% FA as a mobile phase modifier. Though the FA salt form had better aqueous solubility than the free base, it still required 15% dimethyl sufoxide (DMSO) in PBS to observe visible proton signals in te solid . Comparite solid  showed t1H-NMR signals of the ligand instead of the complex protein spectrum. This technique is based on the transfer of saturation from the protein to the bound ligand when the protein is selectively irradiated at a frequency where only resonances from the protein are located. The fast exchange of ligands with the binding sites allows for identification of the epitope of the protein surface close to the bound ligand. The STD-NMR of NUCC-0200590 bound to TRIP8b was initially carried out using a 20-fold ligand excess with respect to the protein at on-resonance frequency of −1 ppm and off-resonance frequency of 40 ppm. To better define the binding epitope, a 100-fold ligand excess was used (20 μM protein:2 mM ligand) in the STD-NMR experiment . On irradiating a solution of NUCC-0200590 (2 mM) and TRIP8b (20 μM) at the on-resonance and off-resonance frequency, there was unambiguous evidence of saturation transfer to the ligand as shown by the STD-NMR (B). A control STD-NMR experiment of the ligand without the protein showed no ligand signal as there was no saturation transfer occurring due to the absence of TRIP8b (C). This confirmed the ability of NUCC0200590 to bind directly to TRIP8b. To get a structural understanding of the binding mode, the relative degree of saturation of the protons in the ligand was analyzed. Integration of the STD-NMR protons normalized to the protons at 7.65 ppm shows the aromatic protons with a 1:1 integral ratio in both the reference spectrum and the STD-NMR spectrum, which indicates that these protons are in close proximity to the protein. In comparison, integration of the aliphatic protons displayed integral ratio from 2:1 to 13:1 (reference spectrum: STD-NMR spectrum). This suggests that these protons were further away from the protein surface in comparison to the aromatic protons. Interestingly, the aliphatic protons of the piperazine showed no saturation transfer as they were not visible in the STD-NMR, indicating that they are not close to the binding pocket of TRIP8b, which agrees with the docked pose of NUCC-0200590 that shows this portion oriented toward the solvent. However, the methyl group on the piperazine showed significant evidence of saturation transfer, suggesting that the methyl group is near the protein surface possibly due to its angle of projection from the piperazine (D). Furthermore, the inhibitor was screened for in vitro stability toward mouse liver microsomes to evaluate its potential metabolism and suitability for future in vivo experiments. The microsomal stability was measured after a 60 min incubation at 37 °C with mouse liver microsomes. NUCC-0200590 was rapidly metabolized by mouse liver microsomes as only 3.7 % of the parent compound remained after incubation.To validate and gain structural understanding of the binding mode of NUCC-0200590 to TRIP8b, we performed STD-NMR experiments on the compound in the presence of TRIP8b. STD-NMR is a popular ligand-observed NMR technique used in studying protein–ligand interactions of weak affinity ligands (high nanomolar to low nanomolar) range. It is a robust technique because it relies on the measurement of relatively simple periment . The refe ligand A. On irr STD-NMR B. A contf TRIP8b C. This cperazine D. Furthein vitro  to confirm our results for both subunits in the experiments later. Thus, the phrase “HCN channels” refers to HCN1 and HCN2 channels collectively.We next turned our attention to testing the ability of NUCC-0200590 to disrupt the TRIP8b–HCN interaction in cell-based assays (using the free base version of the compound for the remainder of the paper). There are many isoforms of TRIP8b that differ in terms of the N-terminal exons that are included (with each isoform named by which exons are included ). As an in vitro , both arin vitro  and haveA). To study the ability of NUCC-0200590 to disrupt the TRIP8b–HCN interaction using full-length proteins, we first employed a NanoBiT split luciferase assay using full-length HCN1 and TRIP8b fused to the relevant Nanoluciferase protein fragments . HEK293T cells were cotransfected with SmBiT-HCN1 and LgBiT-TRIP8b constructs, then pretreated for 30 min with varying concentrations of NUCC-0200590 before adding substrate and reading the luminescent signal. Increasing concentrations of NUCC-0200590 led to a reduction in HCN1–TRIP8b binding as indicated by normalized luminescence, with an IC50 of 5.62 ± 0.49 μM. To ensure the inhibition was specific, we also examined the interaction of the positive control pair protein kinase A catalytic (PRKACA) with type 2A regulatory subunit (PRKAR2A). In the presence of similar concentrations of NUCC-0200590, no change in the binding of these two proteins was observed , consistent with a specific effect of NUCC-0200590 on the TRIP8b–HCN interaction.Having validated NUCC-0200590 as an inhibitor of the TRIP8b–HCN interaction in biophysical and biochemical assays, we tested its effects in several cellular assays. We first examined potential cytotoxicity by incubating HEK293T cells for 3 h or 24 h and analyzing cell viability. No cell death was observed after 3 h of incubation at all tested concentrations of NUCC-0200590 (data not shown). However, after 24 h incubation, at 25 μM and above, we noted cell death, but at lower concentrations there was no effect observed for NUCC-0200590 on cell viability A. To stu50 of Ih by ∼10 mV (h current density) . However, in the presence of 2.5 μM (and higher concentrations) of NUCC-0200590, there was no difference in the V50 between the TRIP8b condition and the control condition (E). Combined, these results indicate that NUCC-0200590 disrupts the hyperpolarizing effect of TRIP8b on the V50 of HCN channels and suggests that NUCC-0200590 causes TRIP8b to dissociate from HCN channels.To test whether the inhibition of TRIP8b–HCN binding by NUCC-0200590 influences HCN channel function, we performed whole-cell recordings using HEK293 cells stably expressing HCN2 . In thesy ∼10 mV , 25, 29,density) , 25, 29.density)  were tradensity)  A-D. Tweior work , 25, 29,ior work E. Howeveondition E. Combinh current density in the absence of NUCC-0200590 (F), but incubation with increasing concentrations of NUCC-0200590 led to a dose-dependent reduction in Ih (F). Combined, these results establish that NUCC-0200590 abolished the known hyperpolarizing effect of TRIP8b on the V50 of HCN channels (suggesting TRIP8b is unbound from HCN) and disrupted TRIP8b-mediated increases in surface HCN channels (indicating fewer surface HCN channels).TRIP8b also upregulates HCN channel current density by increasing HCN channel surface trafficking , 25, 39.-0200590 F, but inon in Ih F. CombinG)  tag was used for flow cytometry such that surface expression of the channel can be measured in live cells . As has Fig. 4G) . Consist50) and surface trafficking, we next asked if these two effects are further dissociable. Specifically, we reasoned that looking at the time course of the change in V50 (TRIP8b binding to HCN channels) and Ih current density (surface expression of HCN channels) could inform the mechanism of NUCC-0200590’s action. Toward that end, we examined the effect of incubating 5 μM NUCC-0200590 on cells stably expressing HCN2 and transiently transfected with TRIP8b. We examined four time points: <1 h, 1 to 4 h, 8 to 10 h, and >24 h. Although it took at least 24 h for the current density (surface HCN channels) to become statistically different from the <1 h incubation condition, (H), the effect on V50 (indicating TRIP8b dissociation from HCN) was apparent at 1 to 4 h (I). These results indicate that NUCC-0200590 first disrupts the effect of TRIP8b on the V50 of Ih (within 1–4 h) prior to the loss of HCN channels from the cell surface. The implications of these findings for the mechanism of NUCC-0200590 are discussed later.After noting that NUCC-0200590 disrupts the effect of TRIP8b on both HCN channel opening , knowing that slices are harder to penetrate and that they cannot be incubated in vitro for long as for HEK cells. We prepared slices from WT mice and incubated them for at least 2 h in 15 μM NUCC-0200590 before whole-cell recordings were made from CA1 pyramidal neurons. In the WT slices, incubation with NUCC-0200590 led to a reduction in the Ih sag ratio, a current clamp measurement of the activity of HCN channels (A/B). There were no differences noted in membrane resistance, resting membrane potential (E). To confirm that NUCC-0200590 limits the sag ratio by binding TRIP8b, we performed a control experiment using slices from genetic KO mice lacking all TRIP8b isoforms (Trip8b−/−) .Having seen that NUCC-0200590 is effective channels A/B. Therotential , or exciotential E. To conin vivo. TRIP8b plays an essential role in trafficking HCN channels into the distal dendrites of CA1 pyramidal neurons, and loss of TRIP8b′s function leads to fewer HCN channels in the distal stratum lacunosum moleculare (A and B).With data validating the effects on NUCC-0200590 in mouse brain slices, we next turned our attention to the question of whether NUCC-0200590 would disrupt TRIP8b function leculare . To circleculare . Compareleculare , A and B−/− mice. Importantly, although these animals lack TRIP8b-mediated HCN channel trafficking, they still express HCN channels in the distal dendrites, and these channels can be further reduced by expressing a TRIP8b mutant after viral manipulation .To confirm that the mechanism for the reduction in HCN1 staining was disruption of the TRIP8b-mediated interaction, we performed the same experiment in Trip8bpulation . In contnjection , C–E.in vitro and in vivo. Direct binding of the compound to TRIP8b was established using STD-NMR, which also provides epitope mapping to show areas of the molecule that closely bind to the target protein . While this result indicates that TRIP8b is likely dissociated from HCN (D), the channel remains present at the cell surface . TRIP8b(1a-4) binding limits lysosomal degradation of the channel (E). Alternatively, it may be the case that the presence of NUCC-0200590 in complex with TRIP8b and HCN could promote an aberrant conformation of the TRIP8b–HCN channel complex that leads to degradation. Future efforts to characterize how NUCC-0200590 limits TRIP8b-mediated surface trafficking of HCN channels could yield a temporally precise method for disrupting HCN channel function that genetic knockdown strategies are not able to achieve.Our cell-based assays demonstrated that NUCC-0200590 effectively disrupts the TRIP8b–HCN interaction and provides an important mechanistic insight into how the compound limits TRIP8b-mediated HCN channel surface trafficking  and 7. Wfrom HCN , C and Dfrom HCN D, the ch channel , 15, 39, channel E. Alternin vivo  and referenced to residual protonated solvent. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s (broad singlet). TLC was performed on glass backed Merck silica gel 60 F254 plates, column chromatography was performed using KP-SIL silica gel (Biotage), and flash column chromatography was performed on Biotage prepacked columns using the automated flash chromatography system Biotage Isolera One. Low resolution LC-MS was performed on a Waters Acquity-H UPLC/MS system with a 2.1 mm × 50 mm, 1.7 μm, reversed phase BEH C18 column and LC-MS grade solvents. A gradient elution from 95% water + 0.1% FA/5% acetonitrile + 0.1% FA to 95% acetonitrile + 0.1% FA/5% water + 0.1% FA over 2 min plus a further minute continuing this mixture at a flow rate of 0.85 ml/min was used as the eluent. Total ion current traces were obtained for electrospray ionization (ESI), positive and negative ionization (ESI+/ESI-). The purities of all the final compounds were of >95% as determined by ultra-performance liquid chromatography analysis unless otherwise indicated. High-resolution mass analysis was performed on Agilent 6210A LC-TOF. Preparative HPLC purification was carried out using a Gilson GX-271 preparative scale reverse phase HPLC system with Gilson model 159 UV–VIS detector and Phenomenex Kinetex 5 μm, C18, 100 A, 50 mm × 30 mm column. Compounds were eluted using a gradient elution of A:B (90:10–0:100) over 5 min at a flow rate of 50.0 ml/min, where solvent A was H2O (with 0.1% FA) and solvent B was CH3CN (with 0.1% FA). The product fractions were combined and dried using a Genevac EZ-2 Centrifugal Evaporator.Unless otherwise noted, all materials for the synthetic chemistry portion were obtained from commercial suppliers  and used without further purification. The The compound ethyl 2-(4-bromo-2-formylphenoxy)acetate was synthesized according to Org. Lett., 2015, 17 , pp 58243, and extracted with DCM (10 ml × 3). The organic layer was dried with Na2SO4, concentrated, and was purified by Biotage eluting with 6% MeOH in DCM to afford 4  as a colorless oil. 1H NMR  δ 7.26 , 7.01 , 6.37 , 4.35 , 3.98 , 3.34 , 2.58 to 2.26 , 2.26 to 2.08 , 2.03 , and 1.02 . 13C NMR  δ 168.41, 155.15, 132.98, 130.37, 129.73, 114.07, 113.50, 65.92, 61.21, 55.40, 55.09, 52.89, 45.95, and 14.10. LC-MS (ESI) m/z: [M + H]+ 371.3.A mixture of ethyl 2-(4-bromo-2-formylphenoxy)acetate , 1-methylpiperazine , and acetic acid  in 1,2-dichloroethane (10 ml) was stirred at RT for 30 min, then sodium triacetoxyhydroborate  was added and stirring was continued for 5 h. The reaction was diluted with dichloromethane (DCM), quenched with NaBHCOin vacuo to yield a crude solid that contained the lithium salt. The crude solid was dissolved in DCM, filtered, and the filtrate was purified by prep HPLC and concentrated to afford 5  as a white fluffy solid. 1H NMR  δ 10.56 , 8.29 , 7.33 , 7.22 , 6.74 , 4.50 , 3.50 , 3.23 to 2.77 , 2.79 to 2.64 , and 2.61 . 13C NMR  δ 172.28, 166.94, 155.23, 134.55, 132.42, 127.20, 114.04, 113.95, 67.19, 57.15, 52.86, 50.25, and 43.26. LC-MS (ESI) m/z: [M + H] + 343.3.To a solution of ethyl 2-(4-bromo-2-((4-methylpiperazin-1-yl)methyl)phenoxy)acetate  in tetrahydrofuran (6 ml) and water (2 ml) was added lithium hydroxide monohydrate , and the reaction was stirred at RT until complete while monitored by TLC. The solvent was removed N-ethyl-N-isopropylpropan-2-amine . The reaction was stirred at RT overnight. The reaction was quenched with water and extracted with ethyl acetate . The organic layers were combined, washed with brine, dried with Na2SO4, and concentrated in vacuo. The crude was purified by prep HPLC and concentrated to afford 6  as a yellow solid. 1H NMR  δ 8.24 , 7.46 , 7.36 , 7.33 to 7.26 , 6.99 , 6.88 to 6.81 , 4.73 , 4.62 to 4.53 , 3.49 , 2.65 to 2.41 , and 2.41 to 2.04 . 13C NMR  δ 168.89, 155.09, 136.79, 134.55, 133.13, 132.01, 128.81, 128.22, 113.92, 113.45, 67.19, 58.53, 54.80, 53.47, 45.93, and 41.95. LC-MS (ESI) m/z: [M + H] + 457.3.To a solution of 2-(4-bromo-2-((4-methylpiperazin-1-yl)methyl)phenoxy)acetic acid  and (4-chlorophenyl)methanamine  in dimethylformamide (4 ml) was added 2--1,1,3,3-tetramethyluronium tetrafluoroborate  and 2CO3  in dioxane (2 ml) and water (1 ml) was added PdCl2(dppf)  and degassed with N2 for 5 min. The reaction vial was sealed and heated to 100 °C for 1 h. The reaction was cooled to RT and extracted with EA (5 ml × 3). The organic layers were combined, washed with brine, dried with Na2SO4, and concentrated in vacuo. The crude was purified with Biotage eluting with 50% EA in hexanes to afford NUCC-0200590  as a thick yellow oil. 1H NMR  δ 7.63 to 7.54 , 7.29 to 7.25 , 7.21 to 7.17 , 7.15 , 7.10 , 4.78 , 4.47 , 3.89 , 2.98 to 2.77 , 2.77 to 2.58 , and 2.45 . 13C NMR  δ 171.45, 163.67 , 157.23, 138.50, 137.66 , 135.29, 134.05, 131.54, 130.01, 129.64, 129.51 , 129.34, 116.57 , 114.27, 68.53, 57.72, 54.42, 52.61, 44.88, and 43.01. HRMS m/z calculated for C22H29F3N4O [M + H]+: 481.1940; found: 481.1937.To a solution of 2-(4-bromo-2-((4-methylpiperazin-1-yl)methyl)phenoxy)-N-(4-chlorobenzyl)acetamide , (4-fluorophenyl)boronic acid , and K2 of NUCC-0200590 was subjected to purification by prep HPLC and concentrated to give the FA salt. 1H NMR  δ 8.52 , 7.61 to 7.53 , 7.26 , 7.18 , 7.14 , 7.10 to 7.06 , 4.77 , 4.47 , 3.86 , 2.92 to 2.76 , 2.76 to 2.60 , 2.44 . 13C NMR  δ 171.42, 163.74 , 157.21, 138.51, 137.68 , 135.25, 134.04, 131.51, 129.98, 129.64, 129.50 , 129.26, 125.87, 116.56 , 114.22, 68.54, 57.72, 54.44, 52.57, 44.82, and 42.98.The crude of the free NH1H NMR  δ 7.76 , 7.70 to 7.63 , 7.33 to 7.29 , 7.29 to 7.24 , 7.19 , 4.96 , 4.48 , 4.43 , 3.78 to 3.29 , and 2.96 . 13C NMR  δ 170.53, 162.97, 161.02, 155.36, 136.58, 134.77 , 133.64, 132.14, 131.21, 129.89, 128.60, 128.21, 128.08 , 117.74, 115.49 , 113.02, 67.09, 55.79, 50.09, 48.50, 42.84, and 42.04.To a solution of NUCC-0200590-free base  in dioxane was added 4M HCl in dioxane . The solution was stirred at RT for 1 h. The solution was concentrated and triturated with ether to afford NUCC-0200590   as a white solid. c40 protein  was incubated with 200 nM of His-tagged TRIP8b (241–602) and varying concentrations of the test compound as described in our prior report . The prepared solution was vortexed for 30 s and submitted for NMR. The epitope mapping experiment was performed the same way, except using 2 mM NUCC-0200590 and 20 μM TRIP8b to increase signal to noise. STD-NMR experiments were performed with a train of 50 ms Gaussian-shaped saturating pulses at 200 Hz power for 2 s with “on” resonance saturation at −1 ppm and “off” resonance saturation at 40 ppm. The relaxation delay was 2 s before the saturating pulses. The number of scans was 2048, and the spectral width was 10 ppm.STD NMR spectrum was recorded using a Bruker Advanced 500 spectrometer equipped with a Cryogenic probe and processed by Bruker Topspin software. An experiment to confirm TRIP8b binding of the inhibitor consisted of compound NUCC-0200590 (100 μM) and TRIP8b(1a-4) (5 μM) in 5% DMSO-dHEK293T cells (ATCC) were maintained as previously described . These cHEK293T cells (obtained freshly from ATCC) were plated in Dulbecco's modified Eagle's medium  with 10% fetal bovine serum , and Pen/Strep  at one million cells per well in 6-well tissue culture–treated plates. The next day, cells were cotransfected using Lipofectamine 3000 with plasmids encoding the small Nanoluciferase fragment (SmBiT) fused to the N terminus of full-length HCN1 (SmBiT-HCN1) and the large Nanoluciferase fragment (LgBiT) fused to the N terminus of TRIP8b(1a-4) (LgBiT-TRIP8b). The plasmids were constructed by cloning into backbone vectors from the Promega NanoBiT MCS cloning kit #N2014. As a control for specific inhibition, other cells were cotransfected with SmBiT-PRKACA and LgBiT-PRKAR2A positive control plasmids from the kit. Twenty-four hours post-transfection, the cells were trypsinized with TrypLE , washed, and suspended in phenol red-free Opti-MEM I , and 25,000 cells per well were plated in white 384-well tissue culture–treated plates . A dilution series of NUCC-0200590 or DMSO vehicle control was added to each well and incubated for 30 min at 37 °C. Luminescence was generated by adding Nano-Glo live cell substrate  and incubating for 30 min at 37 °C prior to detecting on a luminescent reader (Biotek Neo2).The surface HCN expression evaluation by flow cytometry was performed as described previously . HEK293 2, 1 MgCl2, buffered to pH 7.4, and an osmolarity of 312 to 315 mOsm. Intracellular pipette solution contained 135 K-gluconate, 10 MgCl2, 0.1 CaCl2, 1 EGTA, 10 Hepes, and 2 Mg-ATP buffered to pH 7.3 and an osmolarity of 295 to 305 mOsm. Maximal tail current amplitudes were fitted to the Boltzmann equation as described previously , a MultiClamp 700A amplifier (Molecular Devices), and a National Instruments USB6221 interface card. Sampling rate was 10 kHz. Series resistance was monitored throughout each experiment and cells were discarded if the series resistance rose during the experiment. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test.HEK cells stably expressing HCN2 (described in our previous report ) were pleviously . Maximaleviously . Current−/− mice were bred as heterozygotes on a C57BL/6J background as previously described  with a 10 s interval between steps. Data were collected with a Multiclamp 700B amplifier (filtered at 2 kHz) and a Digidata 1440A (digitized at 20 kHz) (Molecular Device Inc) and analyzed with Clampfit 10.0 (Molecular Device Inc).Whole-cell recordings from CA1 pyramidal neurons were performed using an electrophysiology rig described previously . Three hThe stereotaxic injections were performed with minimal modifications from our previous work . Mice ag10.13039/100000002NIH grants  and then analyzed using custom-written routines in MATLAB (Mathworks). The analysis of immunohistochemical images was carried out identically to our previous report (stratum oriens and stratum pyramidale. A larger ROI was also drawn encompassing the stratum lacunosum moleculare and stratum radiatum, which was then subdivided into ten equally spaced ROIs to examine the distal dendritic enrichment of HCN channels.Immunohistochemistry was performed as described in a prior report  using prs report , and a bopen circles indicate individual data points while filled circles represent the mean of that experimental condition.Statistical tests are identified throughout the article with ∗ indicating a statistically significant result. Error bars represent SD and BioRender.comSchematic of TRIP8b-mediated HCN channel trafficking was created using All data described are located in this article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Clinicians, researchers, and patients alike would greatly benefit from more accessible and inexpensive biomarkers for neural β-amyloid (Aβ). We aimed to assess the performance of fully automated plasma Aβ immunoassays, which correlate significantly with immunoprecipitation mass spectrometry assays, in predicting brain Aβ status as determined by visual read assessment of amyloid positron emission tomography (PET).The plasma Aβ42/Aβ40 ratio was measured using a fully automated immunoassay platform (HISCL series) in two clinical studies . The discovery and validation sample sets were retrospectively and randomly selected from participants with early Alzheimer’s disease (AD) identified during screening for the elenbecestat Phase 3 program.We included 197 participants in the discovery study  and 200 in the validation study . The plasma Aβ42/Aβ40 ratio predicted amyloid PET visual read status with areas under the receiver operating characteristic curves of 0.941  and 0.868 (95% CI 0.816–0.920) in the discovery and validation studies, respectively. In the discovery study, a cutoff value of 0.102 was determined based on maximizing the Youden Index, and the sensitivity and specificity were calculated to be 96.0% (95% CI 90.1–98.9%) and 83.5% (95% CI 74.6–90.3%), respectively. Using the same cutoff value, the sensitivity and specificity in the validation study were calculated to be 88.0% (95% CI 80.0–93.6%) and 72.0% (95% CI 62.1–80.5%), respectively.The plasma Aβ42/Aβ40 ratio measured using the HISCL series achieved high accuracy in predicting amyloid PET status. Since our blood-based immunoassay system is less invasive and more accessible than amyloid PET and cerebrospinal fluid testing, it may contribute to the diagnosis of AD in routine clinical practice.The online version contains supplementary material available at 10.1186/s13195-022-01029-0. Alzheimer’s disease (AD) is the most common neurodegenerative disease worldwide . It placMany disease-modifying therapies (DMTs) targeting Aβ have been studied as treatment for AD –9. Recen1-42 (Aβ42) to Aβ1-40 (Aβ40) ratio (Aβ42/Aβ40) has the potential to predict Aβ status as defined by amyloid PET or CSF testing  due to AD and mild AD) according to the core clinical criteria established by the NIA-AA2011 diagnostic guidelines , 32. TheOf these participants, we randomly selected 200 plasma samples for the discovery and validation studies as two different sample sets from participants who underwent amyloid PET to confirm Aβ status. Plasma samples were collected during the screening phase of the trials. The samples were selected to yield approximately 50% positive prevalence of amyloid PET results defined by PET visual read, under the conditions of having both 25% of Japanese participants and 20% of participants with mild AD in each sample set.All studies were conducted in accordance with the Declaration of Helsinki and after approval of the local ethics committees of the participating centers. All participants  provided written informed consent prior to enrolling in MissionAD.g for 10 min. After centrifugation, plasma (0.5 mL) was transferred into 2.0-mL Protein Lobind tubes . Within 30 min of plasma separation, the plasma samples were transferred to a freezer (below − 70 °C).Blood samples were acquired by completely filling a 6-mL plastic K2 EDTA tube . Within 30 min of blood collection, samples were centrifuged at 2000 × The plasma Aβ40 and Aβ42 levels were measured using the HISCL series. The Aβ assays required 30 μL of plasma, and measurements were completed within 17 min per assay. These assay procedures have been previously described .To evaluate the clinical performance of our immunoassay, plasma Aβ42/Aβ40 was used. A cutoff value was determined by maximizing the Youden Index in the discovery sample set. The sensitivity, specificity, positive-predictive value (PPV), and negative-predictive value (NPV) were calculated in both the discovery and validation studies using the same cutoff value.n = 3,492) [Three amyloid PET probes  were used, depending on their availability. The brain Aβ status was centrally assessed by a radiologist in a blinded manner to cognitive status, using the visual read method developed for each PET probe. All scans were independently analyzed by readers trained with respect to the guidelines established by the manufacturer. Centiloids (CL) were calculated by Bioclinica (imaging CRO) using their own CL imaging pipeline, based on the methods described in Klunk et al. (2015) , 34. For= 3,492) .https://analyse-it.com/). Differences between the discovery and validation sample sets were evaluated using the Student’s t-test for continuous measures and the χ2 test for categorical variables. To assess the overall performance in predicting amyloid PET status, the area under the ROC curve (AUC) was obtained by performing ROC analysis using logistic regression. The DeLong method was used to calculate 95% confidence intervals (CI) and to compare the difference in AUCs [P values < 0.05 were considered significant.Statistical analyses were conducted using StatFlex version 7.0 software  and Analyse-it version 5.90 software , 83.5% (95% CI 74.6–90.3%), 85.7% (95% CI 77.8–91.6%), and 95.3% (95% CI 88.4–98.7%) in the discovery study  and 0.868 (95% CI 0.816–0.920), respectively   and 0.922 (95% CI 0.883–0.961) in the discovery and validation studies, respectively .The correlation between plasma Aβ42/Aβ40 levels and CL was also evaluated Fig. . In bothIn the discovery study, 97.5% (79 of 81 samples) of amyloid PET-positive samples, as determined by CL (CL-positive), were also plasma Aβ42/Aβ40-positive. Only two CL-positive samples (2.5%) were categorized as negative for plasma Aβ42/Aβ40. Regarding the amyloid PET-negative samples, as determined by CL (CL-negative), 80.8% (80 of 99 samples) were concordant with plasma Aβ42/Aβ40 results. The remaining 19.2% (19 of 99 samples) were plasma Aβ42/Aβ40-positive. Similar trends were identified in the validation study using the same cutoff values. Overall, 94.5% of CL-positive (86 of 91) and 78.0% of CL-negative (78 of 100) samples were concordant with plasma Aβ42/Aβ40 results. However, 5.5% of CL-positive (5 of 91) and 22% of CL-negative (22 of 100) samples had discordant plasma Aβ42/Aβ40 findings.In this study, we described the clinical performance of fully automated immunoassays using the HISCL series to measure the plasma Aβ42/Aβ40 ratio. Our immunoassay system achieved high accuracy in predicting brain Aβ status, defined via visual reads of amyloid PETs, with AUCs of 0.941 (discovery study) and 0.868 . Our immunoassay system achieved 96.0% sensitivity and 83.5% specificity in the discovery study and 88.0% sensitivity and 72.0% specificity in the validation study. This indicated that the plasma Aβ42/Aβ40 ratio determined using our immunoassay demonstrated a high predictive performance for detecting the brain Aβ status.Several reports have shown that a decrease in the CSF Aβ42/Aβ40 ratio is related to Aβ pathology in the brain, suggesting that the CSF Aβ42/Aβ40 ratio is a promising biomarker for Aβ pathology , 37, 38.IP-MS-based assays for plasma Aβ40 and Aβ42 have shown that a decrease in the plasma Aβ42/Aβ40 ratio has high accuracy in predicting brain Aβ pathology , 22. GivAPOE ε4 is widely considered a major risk factor for the development of AD [APOE ε4 status increases the performance of plasma Aβ42/Aβ40 to predict brain Aβ status [APOE ε4 status; however, this was not significantly different, suggesting that our assay may have high performance, possibly even without including APOE ε4 status.nt of AD . Previouβ status , 42. In The Centiloid method, a standardized quantitative measurement scale, enables comparisons across different amyloid PET probes and sites on the same scale. In light of these advantages, other research groups recently calculated the CL and assessed the performance of blood-based biomarkers for assessing amyloid PET status , 43–45. However, we observed a number of disagreements between the plasma Aβ42/Aβ40 and CL results. These disagreements were mostly observed in the bottom-left area of Fig. In light of the first potential disease-modifying treatment for AD becoming available in the USA, confirming Aβ pathology in patients is becoming increasingly important. Given that the current targets of these drugs are patients with early AD, our immunoassay system could enable the confirmation of brain Aβ status and may facilitate access to treatment for appropriate patients.This study was limited in that, although the two sample sets for the discovery and validation studies were completely independent, they were derived from the same clinical program. Moreover, the discovery and validation studies were conducted in a population with a clinical diagnosis of MCI due to AD or mild AD. These results should be confirmed in different cohorts with more diverse populations, including cognitively unimpaired subjects, racially diverse populations, participants with other neurodegenerative diseases, and real-world, non-clinical trial populations.In conclusion, we developed a fully automated plasma Aβ immunoassay that can predict brain Aβ status with high accuracy. Since aducanumab has already been approved in the US, and since Biologics License Applications submissions of other potential anti-Aβ antibodies have already been initiated, more widely available tests to confirm brain Aβ deposition that are less expensive, less invasive, and easily accessible would reduce the burden on patients and health care providers. Further clinical research using different samples is required. However, this assay may have the potential to fundamentally contribute to the pathophysiological diagnosis of AD in clinical practice.Additional file 1: Table S1. Logistic regression model of plasma Aβ42/Aβ40 ratio combined with APOE ε4 status in the prediction of amyloid PET status. Figure S1. Logistic regression model of plasma Aβ42/Aβ40 ratio combined with APOE ε4 status in the prediction of amyloid PET status. Figure S2. Performance of plasma Aβ levels in the prediction of amyloid PET status determined by Centiloid unit.


Results have been implemented with additional data, which have been commented in the following paragraphs.Enterobacterales ranged from 1 to 8 µg/mL, so zidebactam 0.5 µg/mL, which was a sub-inhibitory dose for all strains tested, was used for testing in combination with aztreonam.” [Results—Checkerboard assays—Section 2.2.1:In Section 2.2.1, The sentence: “In contrast, MIC values for the six Enterobacterales ranged from 1 to 8 µg/mL, so for testing in combination with aztreonam, zidebactam was used both at 0.5 µg/mL, which was a sub-inhibitory dose for all strains tested, and at a 1:1 ratio.”“In contrast, MIC values for the six C. amalonaticus N18, K. pneumoniae KL 12 SG and S. maltophilia but not against the three E. coli, K. pneumoniae LC954/14, C. indologenes and E. meningoseptica (Table 1).” In Section 2.2.1, At the end of the last paragraph, the following sentence was added: “Checkerboard assays testing the combination aztreonam/zidebactam at a 1:1 ratio confirmed previous results, showing synergistic effect against Results—Time-kill assays with aztreonam/zidebactam 1:1 ratio—Section 2.2.4:In Section 2.2.4, A correction has been made to C. amalonaticus and S. maltophilia, while at 1:1 ratio was already synergistic in K. pneumoniae KL 12 SG with 0.5 mg/L concentration . Results were summarized in Figure S1. In C. amalonaticus synergies were also confirmed with aztreonam/zidebactam at 1:1 ratio of 1 mg/L. In S. maltophilia, although we did not detect synergies with 0.5 mg/L of zidebactam (regardless of aztreonam concentration), the two antibiotics showed synergistic effect with a 4 mg/L concentration, although the regrowth observed at 24 h suggests that this combination needs to be further analyzed in a higher number of S. maltophilia isolates.”“Time-kill assays were also performed with aztreonam/zidebactam at 1:1 ratio against 5 CFU/mL. The combination was considered synergistic when the fractional inhibitory concentration index (FICI) was ≤0.5” was changed to Materials and Methods—MIC evaluation and checkerboard assays—Section 4.5:In Section 4.5, The sentence: “ZID, for which a recommended fixed concentration was not available, was tested in the range 0.125–32 µg/mL, alone and in association with ATM, by checkerboard assay in 96-well microtiter plates on an initial inoculum of 5 × 105 CFU/mL. Besides, as the Clinical Laboratory and Standards Institute (CLSI) in the United States of America recommends that testing of zidebactam in combination with cefepime is carried out at a 1:1 ratio, additional testing of the combination aztreonam/zidebactam at a 1:1 ratio was carried out. The combination was considered synergistic when the fractional inhibitory concentration index (FICI) was ≤0.5.”“Zidebactam (ZID), for which a recommended fixed concentration was not available, was tested in the range 0.125–32 µg/mL, alone and in association with ATM, by checkerboard assay in 96-well microtiter plates on an initial inoculum of 5 × 10C. amalonaticus and S. maltophilia.In the section Supplementary Materials, Figure S1 was added. Figure S1 shows time-kill assays performed with aztreonam/zidebactam at 1:1 ratio against The MICs obtained using the combination aztreonam/zidebactam at a 1:1 ratio have been added in The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original publication has also been updated.