E. coli. CSP screening of 80 commercial antimicrobials demonstrated multiple CS interactions. Further screening of a 6195-member natural product library revealed extensive CS relationships in nature. In particular, we report the isolation of known and new analogues of borrelidin A with potent CS activities against cephalosporin-resistant strains. Co-dosing ceftazidime with borrelidin A slows broader cephalosporin resistance with no recognizable resistance to borrelidin A itself.The rapid emergence of antimicrobial resistance presents serious health challenges to the management of infectious diseases, a problem that is further exacerbated by slowing rates of antimicrobial drug discovery in recent years. The phenomenon of collateral sensitivity (CS), whereby resistance to one drug is accompanied by increased sensitivity to another, provides new opportunities to address both these challenges. Here, we present a high-throughput screening platform termed Collateral Sensitivity Profiling (CSP) to map the difference in bioactivity of large chemical libraries across 29 drug-resistant strains of  E. coli strains to identify multiple CS interactions, including natural products with potent CS activities against cephalosporin-resistant strains.Collateral sensitivity (CS), whereby resistance to one drug is accompanied by increased sensitivity to another, provides new opportunities for antimicrobial drug discovery. Here, Liu et al. screen large chemical libraries across 29 drug-resistant  In the most comprehensive study to date, Murray et al. reported an estimated 4.95 million deaths associated with bacterial AMR globally in 2019 alone3. Chief among the list of AMR culprits are pathogenic strains of Escherichia coli, including third-generation cephalosporin-resistant and fluoroquinolone-resistant strains, which in total account for more than 800,000 associated deaths3. AMR is challenging to address because resistance is an inherent consequence of antimicrobial drug use; even a single long-term infection may result in the emergence of drug resistance4. The ubiquitous and non-compliant overuse of antimicrobials in both clinical and agricultural settings contribute to large scale resistance evolution2. Despite recent attention to AMR, the incidence of multidrug resistant (MDR) infections continues to rise5 and resistance to newer drugs is often observed within a few years of deployment6. In contrast, the rate of antimicrobial drug discovery has declined considerably owing to low profitability and shifting priorities within the pharmaceutical industry8, further exacerbating the AMR crisis.The rise of antimicrobial drug resistance (AMR) worldwide represents one of the most pressing contemporary threats to human health11. CS interactions have been widely observed in both drug-resistant microbes and in cancer12, and are thought to result from trade-offs inherent to the maintenance of pleiotropic drug resistance mutations9. Using laboratory-evolved drug-resistant E. coli strains, several studies have mapped out CS relationships between known antibiotics and have demonstrated their application against clinical isolates9. These include the deployment of two reciprocally CS-active drugs in an alternating drug-cycling regimen, such as cefuroxime and gentamicin in E. coli10, or the use of a primary drug with its CS-active adjuvant in combination. For example, using mecillinam with cefotaxime has been shown to constrain the evolution of a globally distributed extended spectrum β-lactamase13. Most bacterial CS studies to date, however, have been limited to known, commercially available antimicrobials14. Few studies have examined the CS potential of natural products (NPs) from an antimicrobial perspective16, despite the obvious value of NP chemical diversity and biological relevance in antibiotic therapy development.One approach to treating AMR strains is to exploit orthogonal sensitivities to one drug that arise from resistance to another drug, a phenomenon known as collateral sensitivity (CS)17.In the context of drug resistance, both known and novel NPs have the potential for CS activity. This provides an opportunity to re-examine the chemical space occupied by known NPs, which may contribute to the discovery and design of new CS-based antimicrobials. Furthermore, CS-active compounds can improve understanding of complex CS mechanisms, as well as provide additional insight into the associated mechanisms of resistanceE. coli, to map and identify natural products that elicit CS across diverse drug-resistant phenotypes, termed Collateral Sensitivity Profiling (CSP). We first validate this approach using a collection of commercial antimicrobials covering 30 drug classes, revealing both new and previously reported CS relationships. Extrapolating this hypothesis into novel chemical space, we report the screening of a library of 6195 marine Actinobacterial and Burkholderia natural product extract prefractions and the observation of widespread CS interactions in nature. From this screen, we describe the isolation and characterization of the borrelidin family of macrolide NPs, including a new analogue borrelidin P, with specific CS activity against cephalosporin-resistant E. coli but not wildtype (WT). To our knowledge, this is the first instance of a new NP compound discovered on the basis of its CS activity. Follow-up experiments reveal that threonine—tRNA ligase (ThrRS), the known target of the borrelidins, contributes to the observed CS interaction with E. coli strains bearing cell wall biosynthesis mutations. Further investigation suggests that other targets of the borrelidins may also play a role in its CS activity. Finally, we show that co-dosing ceftazidime with borrelidin A slows the emergence of cephalosporin resistance and suppresses existing ceftazidime resistance below key thresholds.This work presents a high-throughput screening platform, based on a panel of 29 isogenic drug-resistant mutants of E. coli MG1655 (K-12). Twenty-six of these strains carry chromosomal resistance mutations and were isolated as spontaneous mutants on various antibiotic-containing agar plates, while an additional three strains were transformed using multidrug-resistant (MDR) plasmids from previously characterized clinical isolates19. Each mutant was sequenced to determine the genetic basis of its resistance, relevance to the clinical literature, and to ensure that it harbored only a single mutant gene, where applicable (Supplementary Table gyrA) or subunit B (gyrB); 4 chromosomal multidrug-resistant (MDR) strains with mutations in one of two multidrug efflux pump pathways (marR or acrR); 3 rifamycin-resistant strains with mutations in bacterial RNA polymerase subunit β (rpoB); 4 cephalosporin-resistant strains with mutations in either cell envelope biosynthesis (envZ) or lipopolysaccharide biosynthesis ; and 8 aminoglycoside-resistant strains with mutations in the cytochrome o oxidation pathway (cyoA), ubiquinone biosynthesis genes , or the 30S ribosomal protein S12 (rpsL). The 3 plasmid-borne MDR strains contain vectors from drug-resistant E. coli  or K. aerogenes (RK2). Together, the target panel covers a broad range of resistance mechanisms, including DNA replication, RNA synthesis, protein synthesis, cell wall biosynthesis, MDR, and plasmid-borne resistance. Approximately 60% of these strains contain mutations that have been previously reported in drug-resistant pathogens, with varying levels of clinical relevance26.To identify CS interactions, a panel of 29 drug-resistant strains covering multiple drug classes was derived from the standard lab model We emphasized single, chromosomal mutants in this target panel as opposed to more clinically relevant mobile resistance cassettes for three main reasons. Firstly, the focus of this study was to explore the prevalence of CS in nature using a first-principles approach; target panel selection prioritizing diversity in drug resistance mechanisms was better aligned with this goal compared to exclusive representation by clinically relevant plasmid-borne β-lactamases. CSP therefore aims to identify a broad range of CS responses to provide broad biological target coverage for large libraries containing diverse chemistries. Secondly, CS screening of complex NP extracts necessitated a simple, minimally-variable genetic background due to the potential for interference. Multi-gene mutants and plasmid-borne resistance vectors contain several points of variability, which together may produce synergistic and/or antagonistic interactions that complicate comparison with the WT strain. Instead, we selected only single gene mutants that would unambiguously connect any observed CS interaction to a specific drug resistance mutation and compound/extract. Thirdly, chromosomal resistance mutations are more genetically stable and therefore more amenable to high-throughput screening. In contrast, plasmid-borne strains may require both retention control (selection antibiotic) and copy control (inducer) to standardize expression, significantly complicating CS screening.600 absorbance. The activity of a given test compound/extract against each of the 29 drug-resistant strains was then used to construct an activity ‘fingerprint’ by standardizing to the activity of the WT strain. Loss of activity in one or more mutant strains compared to the WT signified resistance or cross resistance (CR). Conversely, an increase in activity in one or more mutant strains compared to WT inferred a CS interaction. CSP thus provides a clear and comprehensive examination of the CS properties of any compound library in high throughput.For CSP, we developed a fully automated 384-well microtiter assay protocol that systematically tests each chemical sample against all 30 test strains  between WT and drug-resistant strains  was systematically screened using CSP  for each extract-strain combination were normalized by strain and expressed as a percent difference from the wildtype standard (%GrowthMutant – %GrowthWT), then processed through hierarchical clustering to group extracts by their CS phenotype  displayed CR relationships in at least one drug-resistant strain. A significant portion of the NP library elicited strong resistance from the aminoglycoside-resistant Str4 mutant, while a distinct cluster of extracts experienced CR from several MDR strains , the rifamycin-resistant strains , and one fluoroquinolone-resistant strain  produced strong but variable patterns of both CR and CS across the entire natural product library. Uniformity of response between the four related mutant strains was low but the overall potency, either CR or CS, was high in relation to other strains. Some strains were similarly prone to strong CR and/or CS responses, such as the rifamycin-resistant strain Rif1. In total, 1469 extracts (1249 Actinobacteria and 220 Burkholderia) produced CS activity in at least one drug-resistant strain compared to wildtype.Extensive patterns of both CR and CS were observed in this NP dataset. Out of 6195 total, 631 extracts  from their stock concentrations and rescreened against the full target panel  into 80 discrete subfractions with 0-5 UV ‘peaks’ per fraction. These 28 peak libraries were then rescreened in a tertiary assay against relevant members of the drug-resistant target panel to identify active metabolites. Active subfractions were evaluated by high-resolution mass spectrometry (HRMS) analysis to obtain target masses as candidate leads for isolation.The subset of 72 extracts was further triaged based on CS magnitude and profile diversity to give 28 prefractions for downstream chemical investigation. A high-throughput bioactivity-guided fractionation approach, termed the Peak Library system, was adopted to deconvolute these complex mixtures and to identify the active components. Details regarding the Peak Library system are provided in the methods section and have been described in previous work published by our grouprfaH W4*) and Cef7 (rfaG E289fs) and 2-fold MIC shifts for Cef1 (envZ T402M), Cef8 (envZ P248S), and several additional strains  were isolated from a large-scale culture of the producing organism  and rescreened against members of the target panel to obtain absolute MIC shifts for each strain. Compound 1 displayed the strongest CS activity, with 64-fold and 32-fold decreases in its MIC against cephalosporin-resistant strains Cef6 and Cef7, respectively , suggesting that the CS interaction is specific to the resistance genes within RK2. In congruence with both primary and secondary screening results, 1 remained CS-active against other cephalosporin-resistant strains Cef1 and Cef8, as well as fluoroquinolone-resistant strain Cip2KB (gyrB L509G) and aminoglycoside-resistant strain Kn15 (ubiF Q120*). However, the potency of these CS activities was reduced, with only 2- to 4-fold shifts in the IC50 (instead of full inhibition) depending on the strain  in both prokaryotic and eukaryotic targets34. This inhibition is contingent on an unusual nitrile functional group pendant on the macrocyclic core35. Interestingly, 4 displayed CS activity despite the substitution of a primary amide in place of the nitrile group. To test the hypothesis that the observed CS activity of 1 and 4 was related to bacterial threonine—tRNA ligase inhibition, an ASKA library vector containing thrS was transformed into WT, Cef6, and Cef7 E. coli strains. Induction of ThrRS overexpression, followed by treatment with 1, resulted in a 32-fold increase in its MIC against strains Cef6 and Cef7 compared to uninduced control 35. Our results suggest that additional targets may exist in bacteria as well, and that this activity is magnified in select drug resistance phenotypes.Compound rol Fig. . This exrol Fig. . These r1  were screened together using two-dimensional checkerboard assays against WT, Cef7, and the MDR RK2 strains. The β-lactam antibiotic ampicillin was used as a control for ceftazidime. Resulting absorbance data were normalized as percent growth values, with the combination of the lowest compound concentrations resulting in at least 50% inhibition of growth used to calculate the fractional inhibitory index (FIC). In WT E. coli, ceftazidime in conjunction with 1 produced strong additive interactions (FIC = 0.56) but not synergy . Screening in the cephalosporin-resistant strain Cef7 reproduced the expected CS interaction: decreased activity for ceftazidime but increased activity for 1 . Lastly, we tested the MDR strain RK2 as a CS control that do not possess cell wall resistance mutations; an additive interaction was again observed  and vice versa , thereby producing a synergistic interaction. To that end, ceftazidime and 1), or mupirocin  at their respective IC50 for 10 min. Cells of each strain/treatment condition were harvested, with the vehicle control serving to demonstrate specificity of selection product ion transitions. Following the removal of cell culture supernatants, cells were lysed to permit absolute quantification of internalized compounds by targeted liquid chromatography-mass spectrometry (LC-MS) using multiple reaction monitoring (MRM). LC-MS-based quantification revealed no statistically significant increase in the intracellular concentration of 1 between the three strains . Cultures were passaged daily under increasing concentrations of ceftazidime, based on MIC values. Figure 36 breakpoint for ceftazidime resistance in E. coli (>4 μM). Co-dosing with 1 at 32 μM (1/8th its MIC in WT E. coli) marginally suppressed the average MIC compared to ceftazidime alone and prevented resistance from reaching the EUCAST breakpoint for 12 days. Increasing 1 treatment concentration to 128 μM (its IC50 in WT E. coli) reduced resistance by up to 4-fold compared to ceftazidime alone and stabilized resistance between 1 to 2 μM for up to 12 days. In contrast, co-dosing ceftazidime with norfloxacin failed to significantly suppress the emergence of ceftazidime resistance, reaching the breakpoint as early as the 9th day  but greatly exacerbated cefaclor CR (37-fold MIC), illustrating one of the risks of ineffective co-dosing selection. Norfloxacin treatment furthermore produced mild resistance towards the two fluoroquinolone compounds. Yet, resistance against 1 was not observed for any treatment with 1; a mild CS interaction was instead detected with the fluoroquinolones. These results conclude that borrelidin NPs are capable of suppressing the emergence cephalosporin resistance in E. coli.At the conclusion of the serial passaging experiment, cultures from each condition were subjected to treatment with three cephalosporin drugs , two fluoroquinolones , and 1 Fig. . After 137. In this work, we present a high-throughput screening platform, termed CSP, for the systematic identification of CS-active compounds in large chemical libraries. CSP leverages a diverse single mutant target panel to facilitate annotation of the gene/pathway responsible for resistance, and by extension highlight one-compound-to-one-gene CS interactions. Initial screening of 80 commercial antimicrobials covering 30 drug classes using CSP revealed both known and novel CS relationships  producing MICs 128- and 64-fold lower in the Cef6 (rfaH W4*) and Cef7 (rfaG E289fs) strains compared to WT, respectively. Several studies from the existing literature help to contextualize this finding. First, knockouts of genes responsible for lipopolysaccharide (LPS) biosynthesis (rfa) in E. coli have been shown to increase resistance against β-lactams, including cephalosporin compounds40. These mutants produce a variably truncated LPS core that discourages trimerization of outer membrane porins, which themselves facilitate the entry of small polar drugs. Recently, Wang et al. reported that truncated LPS mutants exhibit decreased transcription and expression of porins OmpF and OmpC, but increased transcription of the β-lactam resistance gene blr39. Second, both laboratory and clinical isolates of β-lactam-resistant E. coli have been found to contain mutations in thrS41, the known target of the borrelidins. Studies have shown that inactivation of ThrRS results in a stringent response that reduces bacterial growth rate, which contributes to the observed resistance phenotype42. In this study, we show that 1 is a concentration-dependent inhibitor of ThrRS that preferentially kills cephalosporin-resistant E. coli (with deleterious mutations in rfa genes) as well as a MDR strain carrying the RK2 plasmid from clinical isolates of K. aerogenes19  with ceftazidime suppressed ceftazidime resistance below the EUCAST36 breakpoint for E. coli (4 μM) over a period of 12 days  were inoculated with a single colony each and grown overnight at 37 °C with 150 rpm shaking in 96-well plates. Each liquid culture was then plated in its entirety on an LB agar plate containing one of the following antibiotics: tetracycline (8.44 μM), chloramphenicol (49.5 μM), rifampicin (60.8 μM), ceftazidime (1.14 μM), gentamicin (7.85 μM), kanamycin (15.5 μM), streptomycin (13.8 μM), or ampicillin (17.9 μM). To ensure collection of independent mutants, 20 replicate plates were generated for each antibiotic, and a single colony was picked from each plate showing growth.Isolation of ciprofloxacin-resistant and nalidixic acid-resistant strains was performed as described above. Additional single-step AMR mutants of 45, with parameters LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:36. FastQC was used to quality check trimmed FASTQ files (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reference-guided assembly was carried out using Bowtie2, with the E. coli K-12 (MG1655) genome as a reference (GenBank NC_000913.2). Mutation effects were predicted using SnpEff46. Strains carrying the multidrug resistant plasmids RK2, pAC29 and pAC30 were generated by CaCl2 transformation, as described below for ThrRS overexpression strains. The IncF plasmids pAC29 and pAC30 were taken from clinical isolates of E. coli PB29 and PB3018, while RK2 is a well-described vector from MDR K. aerogenes isolates47.Whole-genome sequencing was carried out on each AMR mutant. Bacterial cultures were grown overnight in 5 mL LB; DNA was extracted using the BioBasic One-4-All Genomic DNA Mini-prep Kit  according to the manufacturer’s instructions. Sequencing was carried out using the MiSeq platform with Nextera XT DNA library preparation and paired-end 250 basepair reads. Raw FASTQ sequence files were trimmed using Trimmomatic v.0.35E. coli strains were inoculated from glycerol stocks into 3 mL overnight cultures in LB media (Fisher), growing at 37 °C, 200 rpm. Saturated overnight cultures were diluted in LB media according to turbidity to achieve ~5 × 105 CFU/mL of final inoculum density and dispensed (Matrix WellMate) into sterile polystyrene 384-well microplates (Thermo ScientificTM 265202) with a final screening volume of 30 μL. Antimicrobials  at 37 °C, 5% CO2 for 20 h. Post-incubation, OD600 readings were taken at t20. Post-data acquisition, absorbance values were normalized by subtracting t0 values from the corresponding t20 values and then dividing by the average of the difference of the blank and growth controls. The MIC for each compound against each strain was determined as the lowest concentration for which there was 90% inhibition compared to controls. The full screening protocol was repeated three times to obtain three independent biological replicates. The Z’-factor for the antimicrobial CSP screen was ascertained by first calculating the Z’-factor for all four compound plates against each test strain, then averaging across three replicates, and finally averaging again across all thirty strains  followed by a 100% ethyl acetate wash, affording six prefractions per extract. Prefractions were concentrated to dryness in vacuo, resuspended in 1 mL of DMSO, and stored in 96-deepwell plates at −70 °C. Replicates of DMSO stock solutions were reformatted into 384-well plates for screening, which proceeded via the protocol outlined above. Only one biological replicate was acquired due to material limitations of the library. For the natural products screen, the Z-factor was calculated instead of the Z’-factor to better control for the discrepancy in signal between complex extracts and pure antimicrobials. This was achieved by replacing the original negative controls  in the Z’-factor calculation with that of the screening data in its entirety, thereby incorporating sample variability49. The interquartile range method was used to reject outliers and prevent hits from skewing the data. See Supplementary Table Cell culturing, extraction, library preparation, and crude extract fractionation were performed in the following sequence. Actinobacterial strains were isolated from marine sediment collected from the North American west coast including sites in California, Oregon, and Washington Statehttps://software.broadinstitute.org/morpheus/) and hierarchically clustered using Spearman-Rank correlation and average linkage parameters50. 117 hit extracts were selected from this analysis for secondary screening. DMSO library stocks of these prefractions were reformatted into 16 two-fold dilutions in 384-well microplates starting from their stock concentrations. Secondary screening and subsequent data processing were performed as described for the primary NP screen. Twenty-eight prefractions were selected for further chemical investigation from the secondary screen based on the magnitude and reproducibility of their CS profiles.Primary screening data was subjected to plate-based normalization as described above, then normalized again across all plates for each given strain to standardize the maximum growth. Lastly, WT normalized percent growth values for each extract were subtracted from their respective strains’ growth values such that positive values inferred increased resistance while negative values indicated collateral sensitivity. Processed screening data were uploaded to the Morpheus data analysis platform  at RT for up to 5 days. Discrete colonies were used to inoculate 7 mL of SYP liquid media  with glass beads, shaking at room temperature (RT), 200 rpm for 4 days. 3 mL of this liquid culture was then used to inoculate 60 mL of SYP liquid media with a coiled stainless-steel spring, shaking at RT, 200 rpm for an additional 5 days. 45 mL of the previous culture was used to inoculate 1 L of SYP liquid media inside a sterile 2.8 L wide-neck Fernbach flask containing a larger coiled stainless-steel spring and XAD-17 resin (20 g), shaking at RT, 200 rpm for another 7 days. After fermentation, the large-scale culture was filtered with Whatman paper, and the residue extracted with DCM/MeOH (1:1) three times while stirring. The resulting suspension was filtered again to remove cellular debris and torn filter paper. Celite resin (20 g) was added to the filtrate and evaporated to dryness in vacuo. Extract-adhered dry resin was packed into a reusable prep column and prefractionated using the CombiFlash via an eluotropic series of MeOH/H2O as described for NP library preparation. The appropriate fractions were collected separately and concentrated to dryness under vacuum for further analysis. Resulting prefractions corresponding to hits from the secondary screen were rescreened against selected strains to ensure reproducibility of activity. Bioactive prefractions from the fermentation step were then prioritized for subsequent bioassay-guided fractionation via the Peak Library system.Frozen stocks  of each of the 28 producing organisms were plated on solid MB media , and centrifuged to remove particulate. The resulting supernatant was fractionated via HPLC-MS (Phenomonex Kinetix 2.6 µm XB-C18 150 × 4.6 mm) into 80 discrete subfractions in a 96-deep-well microplate. HPLC gradients used for fractionation varies based on the prefractionation process of each extract. Prefraction A (20% MeOH/H2O wash): gradient of MeOH/H2O + 0.02% formic acid  at a flow rate of 1 mL min−1, collecting 1 subfraction every 0.5 min starting at 5 min. Prefraction B (40% MeOH/H2O wash): gradient of MeOH/H2O + 0.02% formic acid  at a flow rate of 1 mL min−1, collecting 1 subfraction every 0.5 min starting at 5 min. Prefraction C (60% MeOH/H2O wash): gradient of MeOH/H2O + 0.02% formic acid  at a flow rate of 1 mL min−1, collecting 1 subfraction every 0.5 min starting at 5 min. Prefraction D (80% MeOH/H2O wash): gradient of MeOH/H2O + 0.02% formic acid  at a flow rate of 1 mL min−1, collecting 1 subfraction every 0.5 min starting at 5 min. Prefraction E (100% MeOH wash): gradient of MeOH/H2O + 0.02% formic acid  at a flow rate of 1 mL min−1, collecting 1 subfraction every 0.5 min starting at 5 min. 96-deep-well microplates containing extract subfractions were concentrated to dryness using a Thermo SpeedVac concentrator . Dry extracts were resuspended in 10 µL of DMSO and reformatted into 384-well compound plates. Screening of peak libraries proceeded as described for the primary and secondary NP screens.For each bioactive extract, a standardized volume of the DMSO stock solution was transferred from storage at −70 °C, evaporated to dryness Streptomyces spp. strain RL09-056-NTS-A) was profiled by UPLC-HRMS using a Waters SYNAPT G2Si ESI-qTOF-MS system running MassLynx software and LC-MS using an Agilent 6130 MS instrument running ChemStation. Four related analogs with UV maxima at ~260 nm and retention times between 10.0 to 12.0 min were prioritized for purification based on its Peak Library screening data. The major component, possessing a m/z of 512.2981 [M + Na]+ was purified by HPLC to yield 9.82 mg of a white amorphous powder. Extensive 1D and 2D NMR analyses  identified this compound as the known macrolide borrelidin A  or the olefin at C14 (3). The fourth bioactive analog eluted earlier than 1 at 10.2 min and possessed a formula that did not correspond to any known borrelidin derivatives54. Consequently, this new molecule was named borrelidin P (4). Interpretation of HRMS and NMR data −14-(E) geometry in the diene region of the natural product (as opposed to a 12-(E)-14-(E) arrangement for 5), completing the planar structure determination for this new metabolite. To assign the absolute configuration of 4, the natural product was derivatized via a series of reactions to facilitate the eventual conjugation of the modified Mosher’s ester on secondary alcohols. Compound 4 was first protected by esterification of the carboxylic acid at C23 to produce the methyl ester-capped intermediate -Mosher’s acid chloride to form the linearized borrelidin P methyl diester (S)-MTPA conjugate -MTPA conjugate was made with the Mosher’s analysis performed instead using the variable temperature method reported by Latypov et al.56 Chemical shift differences for protons adjacent to the secondary alcohols at C3 and C11 were determined to be 3-(S)−11-(R) : white amorphous powder; 1H-NMR : δ 6.92 , 6.61 , 6.33 , 4.98 , 4.19 , 3.94 , 2.67 , 2.59 , 2.53 , 2.40 , 2.24 , 2.02 (overlap), 2.01 (overlap), 1.88 (overlap), 1.86 (overlap), 1.82 (overlap), 1.81 (overlap), 1.78 (overlap), 1.63 (overlap), 1.42 , 1.24 , 1.19 , 1.09 , 1.04 , 0.99 (overlap), 0.97 (overlap), 0.87 , 0.87 , 0.85 , 0.70 ; 13C-NMR : δ 173.3, 145.5, 140.3, 128.9, 119.9, 117.4, 77.5, 73.1, 72.9, 49.4, 47.5, 44.6, 39.1, 37.9, 37.1, 36.7, 35.9, 32.6, 30.5, 28.5, 27.6, 26.2, 20.9, 19.1, 18.7, 15.4; UV/Vis: λmax 260 nm; HRMS (m/z): [M + Na]+ calcd. for C28H43NNaO6, 512.2983; found, 512.2981.Borrelidin A (2): white amorphous powder; 1H-NMR : δ 6.79 , 6.36 , 6.01 , 4.99 , 3.71 (overlap), 3.70 , 2.59 , 2.38 (overlap), 2.38 (overlap), 2.29 , 2.09 , 1.84 , 1.77 (overlap), 1.75 (overlap), 1.68 (overlap), 1.66 (overlap), 1.63 (overlap), 1.59 , 1.52 , 1.35 , 1.22 (overlap), 1.04 , 0.96 (overlap), 0.96 (overlap), 0.91 , 0.81 , 0.77 , 0.74 , 0.74 , 0.68 ; 13C-NMR : δ 144.2, 140.5, 128, 77.1, 74.6, 69.3, 47.8, 47.4, 42.3, 37.5, 36.8, 36.6, 34.8, 34.6, 30.6, 28.7, 26.4, 25.7, 24.8, 20.1, 17.7, 17.7, 15.5; UV/Vis: λmax 260 nm; HRMS (m/z): [M + H]+ calcd. for C28H44NO6, 490.3163; found, 490.3172.Borrelidin F (3): white amorphous powder; 1H-NMR : δ 7.12 , 6.46 , 6.02 , 4.94 , 3.81 , 3.75 , 2.67 , 2.39 (overlap), 2.33 , 2.23 , 2.01 , 1.86 (overlap), 1.86 (overlap), 1.80 , 1.71 (overlap), 1.69 (overlap), 1.65 (overlap), 1.64 (overlap), 1.52 , 1.36 , 1.11 (overlap), 1.08 (overlap), 1.06 (overlap), 0.93 (overlap), 0.91 , 0.88 (overlap), 0.78 (overlap), 0.76 (overlap), 0.75 (overlap), 0.68 ; 13C-NMR : δ 138.3, 135.2, 126.6, 76.4, 74.7, 70.3, 47.9, 47.5, 43, 36.7, 35.2, 34.9, 30.9, 30.6, 28.6, 26.7, 26.1, 24.7, 20.1, 19.1, 18.7, 15.0; UV/Vis: λmax 258 nm; HRMS (m/z): [M + H]+ calcd. for C28H44NO6, 490.3163; found, 490.3170.Borrelidin H (4): white amorphous powder; 1H-NMR : δ 6.72 , 6.19 , 5.82 , 5.06 , 3.84 , 3.74 , 2.57 (overlap), 2.53 (overlap), 2.40 (overlap), 2.36 (overlap), 1.96 (overlap), 1.87 (overlap), 1.85 (overlap), 1.76 (overlap), 1.74 (overlap), 1.74 (overlap), 1.71 (overlap), 1.64 (overlap), 1.63 (overlap), 1.39 (overlap), 1.37 (overlap), 1.14 , 1.07 , 1.03 (overlap), 0.98, , 0.95 (overlap), 0.86 , 0.85 (overlap), 0.85 , 0.82 ; 13C-NMR : δ 183.6, 174.7, 172.6, 137.9, 136.4, 135.5, 130.0, 83.0, 77.7, 71.6, 49.5, 49.0, 44.0, 40.6, 39.1, 38.8, 37.0, 35.9, 33.1, 30.6, 28.1, 27.8, 26.3, 20.8, 19.3, 16.7, 16.5; UV/Vis: λmax 249 nm; HRMS (m/z): [M + H]+ calcd. for C28H46NO7, 508.3269; found, 508.3280.Borrelidin P , acetaldehyde oxime , copper oxide , and MeOH/H2O  were added to a 10 mL conical-bottom flask. The reaction mixture was heated to reflux at 150 °C for 60 h. A small aliquot was removed every 12 h to monitor reaction progress via HPLC-MS. After completion, the reaction mixture was evaporated to dryness in vacuo and resuspended in 100% methanol. The resulting solution was injected onto a 2 g C-18 cartridge and washed with an eluotropic gradient of 20%, 40%, 60%, and 80% v/v MeOH/H2O. The 20% and 40% fractions containing a mixture of borrelidin A reagent and derivatized products were combined and further purified via HPLC to give (5) as a light-yellow amorphous solid : [M + H]+ calcd. for C28H45NO7, 508.3274; found, 508.3267).The following procedure is adapted from Hu et al.4  in DCM/MeOH  was added (trimethylsilyl)diazomethane . The reaction was allowed to proceed for 4 h at RT. After this time, the reaction mixture was evaporated to dryness via a continuous stream of N2 gas to yield 6 : [M + H]+ calcd. for C29H47NO7, 522.3431; found, 522.3426) as an off-white powder. The crude product was used immediately in the subsequent reaction. in MeOH (1 mL) was added NaOMe . The reaction was allowed to proceed for 60 h at RT. After this time, the reaction mixture was quenched with a saturated aqueous solution of NH4Cl (5 mL). The mixture was transferred to separatory funnel and DCM (5 mL) was added. The organic layer was separated, and the aqueous layer was extracted with DCM (3 × 5 mL). The combined organic extracts were dried (Na2SO4), filtered, and solvent was removed in vacuo. The crude product was purified via HPLC-MS (Phenomenex Kinetix 2.6 µm XB-C18 150 × 4.6 mm) using a gradient of MeCN/H2O + 0.02% formic acid  at a flow rate of 1.5 mL min−1. The appropriate fractions were pooled and solvent removed in vacuo to yield 7 : [M + H]+ calcd. for C30H51NO8, 554.3693; found, 554.3693) as an off-white solid. was dissolved in pyridine-d5 (0.5 mL) in a 5 mm NMR tube, and (R)-MTPA-Cl  was added via micro-syringe. The reaction was monitored via 1H-NMR. After 15 min, 1H-NMR showed complete conversion of the starting material to product 8. After this time, the reaction mixture was evaporated to dryness under a continuous stream of N2 gas. The crude product was dissolved in DCM (5 mL) and a saturated aqueous solution of NaHCO3 (5 mL) was added. The mixture was transferred to separatory funnel, and the organic layer separated. The aqueous layer was extracted with DCM (3 × 5 mL). The combined organic extracts were dried (Na2SO4), filtered, and solvent was removed in vacuo to yield crude product 8 : [M + Na]+ calcd. for C60H72F9NNaO14, 1224.4701; found, 1224.4750) as an off-white solid which was used for 1H-NMR variable temperature modified Mosher’s ester analysis. for 2 h. A 100 μL aliquot was plated to selective LB agar containing 92.8 μM chloramphenicol. Individual colonies were picked after 24 h of growth at 37 °C, and plasmid extractions were performed to confirm the presence of the thrS plasmid. Pure samples of 1 and 4 were prepared as 1:1 dilution series  in DMSO and reformatted onto a 384-well plate. Both compounds were screened in triplicate against WT E. coli and Cef6, Cef7, RK2, WT-(thrS), Cef6-(thrS), Cef7-(thrS) mutant strains according to the protocol described for the antimicrobial control panel. For the (thrS)-transformed mutants, one set of diluted cultures were dispensed normally while a second set was treated with 0.1 mM IPTG inducer 1 h prior to dispensation. Resultant growth data was processed and normalized to controls, then visualized as Hill slope curves using GraphPad Prism.WT 1, and ampicillin were prepared as 15 two-fold dilution series on three separate compound plates: 1 was arranged in a horizontal gradient across plate one , then replicated vertically for 15 rows (row A–O); ceftazidime and ampicillin were arranged in vertical gradients across plates two and three, respectively , then replicated horizontally for 15 columns (col. 3–17). Plates one-two and one-three were screened in combination against WT, Cef6, and Cef7 strains using the protocol described above, with the liquid handling procedure modified to pin two compound plates into each assay plate. Resulting growth data was normalized as percent growth values and visualized as two-dimensional heatmaps. The FIC index was calculated by selecting the lowest combination of concentrations for each compound producing at least 50% inhibition in growth. Values less than 0.5 are considered synergistic; values in-between 0.5 and 4 are additive/indifferent, and values greater than 4 are antagonistic.Ceftazidime, 58 using Cef6 and Cef7, with WT E. coli as a control. Overnight cultures of each bacterium were grown in LB Miller Broth, and aliquoted into 0.25 mL samples. Five replicates (n = 5) were prepared for each bacterial strain per drug condition , although one replicate was lost for the Cef6 intracellular borrelidin treatment (n = 4). Each sample was pelleted and washed in 1 mL 1x phosphate buffered saline (PBS) , centrifuged, and resuspended in 0.5 mL 1x PBS. All samples were then equilibrated at 37 °C with shaking (150 rpm) for 5 min. 0.5 mL of 1x PBS containing 8 μM of either 1 or mupirocin was added to each sample for a final drug concentration of 4 μM; 0.5 mL 1x PBS with no drug was used for control samples. Vials were allowed to stand for 10 min at room temperature, then pelleted by centrifugation , with the supernatant (SPNT 1) removed and kept. Pellets were washed in 0.5 mL 1x PBS, centrifuged, and the second supernatant (SPNT 2) was removed and pooled with the first supernatant. Cell pellets were resuspended in 210 μL sterile milliQ dH2O, then lysed through a series of freeze/thaws, consisting of 10 cycles of submersion in liquid nitrogen for 3 min, followed by 3 min in a 65 °C water bath. After cell lysis, 5 μL was removed from each sample for spot plating onto LB agar to ensure the absence of cell growth. The lysate was collected by centrifugation , removing the supernatant (SPNT 3), resuspending samples in 100 μL sterile methanol, centrifuging again, and removing the final supernatant (SPNT 4) and combining with SPNT 3. Samples were stored at −20 °C before MS analysis.Borrelidin accumulation assays were conducted following the protocol from Richer et al.8 2.7 μm, 2.1 × 50 mm column. Solvent A: 5 mM ammonium acetate, Solvent B: acetonitrile. The gradient was 15% Solvent B for 0.5 min, 15–50% solvent B over 2 min, 50–95% solvent B over 0.5 min, held at 95% solvent B for 1.5 min, 95–15% solvent B over 0.5 min, and finally re-equilibrated at 15% solvent B for 3 min. 1 and mupirocin concentrations were determined using two period MRM on a SCIEX QTrap 5500 instrument. Calibration curves were prepared with a range of 0.003 to 5 μM. For 1, the transition 488.3 (Q1) > 271.3 (Q3) was monitored at 3.3 min, using a collision energy of −25 V59. For mupirocin, the transition 501.1 (Q1) > 327.1 (Q3) was monitored at 2.6 min, using a collision energy of 17 V60. MS data were acquired using Analyst v1.6.2 and peak areas were integrated using Skyline v21.1.0.146. 1 and mupirocin concentrations were determined with a linear regression using a (1/concentration) weighting.Quantification of target molecule internalization was achieved by liquid chromatography mass spectrometry (LC-MS) using MRM. RP-HPLC was performed using a Waters CORTECS CE. coli K-12 MG1655 was used to inoculate four separate treatment conditions: treatment A, increasing concentrations of ceftazidime starting at 0.063 μM; treatment B, treatment A plus a constant concentration of 1 at 32 μM; treatment C, treatment A plus a constant concentration of 1 at 128 μM; and treatment D, treatment A with increasing concentrations of norfloxacin starting at 0.016 μM. All treatments are performed in triplicate in 96- well plates with 200 μL of LB media. Four concentrations were tested for each treatment, increasing two-fold from the starting concentration . Post-treatment and inoculation, plates were incubated at 37 °C, 5% CO2 for 24 h before an absorbance reading at OD600 is taken. The highest drug concentration resulting in at least 50% growth inhibition for each replicate was used to inoculate the subsequent day’s treatment wells at a 1:100 ratio. Ceftazidime and norfloxacin drug concentration gradients were also modified in 2-fold steps as required to ensure that at least one concentration that produced 100% growth. Borrelidin A treatments were maintained at fixed concentrations. Resistance monitoring/sequential passaging was repeated daily for 12 consecutive days, the maximum length of time achievable given the high rate of consumption of a limited NP compound. The difference between day 1 and day 12 MIC values for each passaging condition was analyzed by a one-way ANOVA with Tukey’s HSD test. At the conclusion of the experiment, the most resistant samples from each replicate were archived as glycerol stocks at −70 °C. To determine the MIC for each resistant strain under standard conditions, glycerol stocks were thawed and used to inoculate overnight cultures. Ceftazidime, cefadroxil, cefaclor, ciprofloxacin, norfloxacin, and borrelidin A were prepared as 1:1 dilution series (128 μM to ~3.9 nM) in DMSO and reformatted into a 384-well plate. All compounds were screened against the ceftazidime-resistant strains following standard procedures described above and their MICs determined for each resistant replicate.An overnight culture of WT Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Osteoarthritis (OA) is the most prevalent age-related disease in the world. Chondrocytes undergo an age-dependent decline in their proliferation and synthetic capacity, which is the main cause of OA development. However, the intrinsic mechanism of chondrocyte senescence is still unclear. This study aimed to investigate the role of a novel long non-coding RNA (lncRNA), AC006064.4–201 in the regulation of chondrocyte senescence and OA progression and to elucidate the underlying molecular mechanisms.The function of AC006064.4–201 in chondrocytes was assessed using western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) and β-galactosidase staining. The interaction between AC006064.4–201 and polypyrimidine tract-binding protein 1 (PTBP1), as well as cyclin-dependent kinase inhibitor 1B (CDKN1B), was evaluated using RPD-MS, fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down assays. Mice models were used to investigate the role of AC006064.4–201 in post-traumatic and age-related OA in vivo.Our research revealed that AC006064.4–201 was downregulated in senescent and degenerated human cartilage, which could alleviate senescence and regulate metabolism in chondrocytes. Mechanically, AC006064.4–201 directly interacts with PTBP1 and blocks the binding between PTBP1 and CDKN1B mRNA, thereby destabilizing CDKN1B mRNA and decreasing the translation of CDKN1B. The in vivo experiments were consistent with the results of the in vitro experiments.The AC006064.4–201/PTBP1/CDKN1B axis plays an important role in OA development and provides new molecular markers for the early diagnosis and treatment of OA in the future.Schematic diagram of AC006064.4–201 mechanism. A schematic diagram of the mechanism underlying the effect of AC006064.4–201The online version contains supplementary material available at 10.1186/s40364-023-00477-6. OA is the most prevalent degenerative disease and the main cause of disability in older adults worldwide. It has affected 32.5 million adults in the United States, at an annual cost of $136.8 billion a year . With thCellular senescence is a stress response induced by multiple intrinsic and extrinsic stimulators . It is cLong non-coding RNAs (lncRNAs) are a subclass of non-coding RNAs (ncRNAs), with a length of approximately 200 nucleotides . PreviouIn the current study, we identified a novel biomarker (AC006064.4–201) in chondrocytes that was negatively correlated with the processes of chondrocyte senescence and OA development. In terms of mechanism, AC006064.4–201 could inhibit the binding of cyclin-dependent kinase inhibitor 1B (CDKN1B) mRNA to polypyrimidine tract-binding protein 1 (PTBP1), thereby downregulating CDKN1B protein translation. The AC006064.4–201/PTBP1/CDKN1B axis is expected to be a potential target for OA treatment in the future.This study was approved by the Ethics Committee of Sir Run Run Shaw Hospital . Human knee joint samples were obtained from patients of different ages who had undergone total knee replacement surgery, and written informed consent was obtained from each patient. Patients with autoimmune and metabolic diseases such as hyperlipidemia, hypertension, diabetes, rheumatoid arthritis, and other diseases that affect the joints were excluded from this study. Samples were divided into two groups according to the age of the corresponding patients: samples from patients aged 50–65 years were taken as the young group, while samples from patients aged 66–80 years were taken as the old group.2 and 100% humidity.Human cartilage tissues were isolated from human knee joint samples and mouse cartilage tissues were isolated from mice that were 5 days old. Cartilage tissues were shredded using the physical method, washed with sterile phosphate buffered saline (PBS), and treated with 0.2% type II collagenase  for 24 h at 37 °C. The mixture was filtered using a 0.075 mm cell strainer and centrifuged at 1500 rpm for 10 min. Finally, the sediments were cultured evenly in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS , and maintained in an incubator set to 37 °C with 5% COAll animal experiments were approved by the Institute of Health Sciences Institutional Animal Care and Use Committee.The adeno-associated virus (AAV) Gm49317-201 short hairpin RNA (shRNA), CDKN1B shRNA and negative control lentivirus were constructed and packaged by HanBio .For post-traumatic arthritis animal models, forty adult male C57BL/6 mice, aged 12 weeks, were used for in vivo experiments. As a positive control, an OA model was introduced using destabilization of the medial meniscus (DMM) surgery, as previously described . BrieflyFor the natural senescence animal model, 30 adult male C57BL/6 mice, aged 4 months, were used for in vivo experiments. Briefly, the mice were randomly divided into three groups  with 10 mice in each group. A total of 10 μl (approximately 1 × 10^11 PFU/mL) of the control virus, Sh Gm49317-201 virus or Sh CDKN1B virus was injected into the knee joints with a microinjector. The injection was repeated 4 months after the first injection. Four months after the second injection, the mice were sacrificed and the knee joints were separated for micro-CT evaluation and histological analysis.The Antisense Oligonucleotides (ASOs) lAC006064.4–201 and Gm49317-201 were designed and constructed by RiboBio . Lipofectamine RNAiMAX (Thermo Fisher Scientific) was used for ASO transfection, according to the manufacturer’s instructions. The sequences are listed in Supplementary Table Overexpression plasmids and shRNA plasmids of PTBP1 and CDKN1B were designed and constructed by TsingkeBio . Virus vectors and packaging plasmids were co-transfected into HEK-293 T cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. The medium was changed 6 h after transfection. HEK-293 T cells were transfected for 48 h, and the medium was collected, centrifuged at 3000 rpm for 10 min, supplemented with 10 μg/mL polybrene (SolarBio), and added to human or mouse chondrocytes. Finally, the cells were selected using 2 μg/mL puromycin for 24 h.Chondrocytes were lysed with radioimmunoprecipitation assay buffer  containing 100 mM phenylmethanesulfonyl fluoride (PMSF) on ice for 20 min. Protein concentrations were determined by bicinchoninic acid (BCA) analysis . Equivalent amounts of proteins were then separated by sodium dodecyl sulphate-polycrylamide gel electrophoresis (SDS-PAGE) at different concentrations, transferred onto polyvinylidene fluoride membranes (Bio-Rad), blocked with 5% nonfat milk at room temperature for 1 h, and incubated with primary antibody at 4 °C overnight. The following day, the membranes were washed by tris-buffered saline (TBST) and incubated with a secondary antibody at room temperature for 1 h. Finally, the protein bands were visualized using FDbio-Femto ECL  and a chemiluminescence system . The antibodies used in this study are listed in Supplementary Table Total RNA was extracted from primary chondrocytes or cartilage tissues using RNAEX reagent , according to the manufacturer’s instructions. Specific mRNAs were qualified using SYBR® Green Premix Pro Taq HS qPCR kit , according to the manufacturer’s instructions. The levels of lncRNAs and mRNAs were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers used are shown in Supplementary Table Cy3-labeled lncAC006064.4–201 and Gm49317-201 probes were designed and synthesized by HaokeBIO . Probe signals were detected using a FISH kit (RiboBio), according to the manufacturer’s instructions. Nuclei were stained with DAPI. Images were acquired using a fluorescence microscope . The Cy3-labled probes used in this study are listed in Supplementary Table For cell immunofluorescence (IF), chondrocytes were fixed with 4% paraformaldehyde for 30 min and permeated with 0.5% tritonX-100 for 30 min. For tissue IF, cartilage specimens were fixed in 4% paraformaldehyde for paraffin embedding and sectioned at 5 μm. Then, the cells or sections were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h and incubated with primary antibody at 4 °C overnight. After washing with PBS, cells or sections were incubated with CL594- or CL488-conjugated secondary antibodies  for 1 h. Nuclei were stained with DAPI. Images were acquired using a fluorescence microscope . The fluorescence intensities were quantified as previously described . The antChondrocyte senescence was determined using a Senescence β-Galactosidase Staining Kit , according to the manufacturer’s instructions. The percentage of positive cells was calculated using Image-Pro Plus 6.0 .7 HEK-293 T cells were pelleted and resuspended in 100 μL of RIP Lysis Buffer supplemented with a protease inhibitors cocktail and ribonuclease inhibitors. Then, the cell lysates were incubated with antibody against PTBP1 (Abcam) or IgG at 4 °C overnight and treated with proteinase K buffer. Finally, the immunoprecipitated RNA were extracted using a RNeasy MinElute Cleanup Kit (Qiagen) and reverse transcribed . The expression levels of AC006064.4–201 were determined by qRT-PCR.HEK-293 T cells were transfected with PTBP1 plasmid or vector. A Magna RIP RNA-Binding Protein Immunoprecipitation Kit  was used to perform RIP experiments. Briefly, approximately 1 × 107 human or mouse chondrocytes were harvested and lysed. The AC006064.4–201, Gm49317-201 and Oligo probes were added to the magnetic beads. The cell lysates were incubated with these probe-coated beads at 4 °C overnight. The RNA–protein complexes were then eluted for western blotting analysis. The biotinylated probes used in this study are listed in Supplementary Table Biotinylated AC006064.4–201 and Gm49317-201 probes were designed and synthesized by RiboBio . An RNA pull-down kit  were used for RNA pull-down assay. Approximately 1 × 10Mouse knee joint samples were fixed in 70% ethanol and scanned using a high-resolution a high-resolution micro-CT instrument , according to the manufacturer’s instructions. The data were analyzed using Skyscan software.Cartilage specimens were fixed in 4% paraformaldehyde for paraffin embedding and sectioned at 5 μm. The sections were dehydrated and stained with Safranin-O/Fast green , according to the manufacturer’s instructions. The Osteoarthritis Research Society International (OARSI) score was based on safranin O/fast green staining of each specimen, as previous described .p < 0.05 between groups.Statistical analysis was performed using the SPSS version 18.0 software . Data were analyzed using Student’s t-test, Fisher’s exact test, and one-way analysis of variance (ANOVA). The results are presented as the mean ± standard deviation (SD). Group differences were considered statistically different for n = 15), and the medial tibial plateau was compared with the lateral tibial plateau. The results demonstrated that AC006064.4–201 exhibited lower expression in the older group and decreased expression on the medial side of the tibial plateau , we performed RNA-seq analyses on first-passaging and third-passaging chondrocytes and identified ten lncRNAs that were most differentially downregulated with the senescence of HCs Table A. Among INK4a, p21, and p53) and degradation enzymes (Mmp3 and Mmp13) and reduced mRNA levels of Sox9, Aggrecan and Col2a1  that could specifically and stably knock down AC006064.4–201 in HCs were used  –26. To iSubsequently, we explored whether AC006064.4–201 affects PTBP1 expression. However, qRT-PCR analysis and western blotting showed that both the mRNA and protein levels of PTBP1 did not change when AC006064.4–201 was knocked down or overexpressed in the HCs Fig. H and I. PTBP1 was shown to bind to the mRNA of CDKN1B and improve its stability . This waINK4a, p21, p53, Mmp3 and Mmp13, while expression of Sox9, Aggrecan and Col2a1 was increased  / PTBP1 / CDKN1B axis is conserved between humans and mice. Moreover, AC006064.4–201 and Gm49317-201 had similar functions in HCs and MCs. This prompted us to conduct further in vivo experiments using mice.To investigate the role of Gm49317-201 in post-traumatic OA, a mouse model was introduced in this study, as described in the Methods section  family, PTBP1 is a widely studied RNA binding protein that binds to the polypyrimidine sequence on the pre-mRNA, and involves in regulating mRNA splicing, translation, stability and localization , 43. PreTaken together, we evaluated the function of the AC006064.4–201/PTBP1/CDKN1B axis in cartilage senescence and degeneration in this study. However, aberrant subchondral remodeling and synovitis are also crucial aspects of OA progression, which will be explored in future studies.In summary, our research revealed a novel molecular marker, AC006064.4–201, that was found to protect against OA by alleviating senescence and degeneration of cartilage. Mechanistically, AC006064.4–201 could destabilize CDKN1B mRNA by interacting with PTBP1 and decreasing the protein expression of CDKN1B. Cumulatively, this study provides new molecular targets for the early diagnosis and treatment of OA.Additional file 1: Supplementary Figure 1. (A) Expressionof ten lncRNAs in HCs assessed by qRT-PCR after treating with 10ng/ml IL-1β.  * p<0.05, ** p<0.01. (B) Fluorescence intensity of FISH staining forAC006064.4-201 and IF staining of p16INK4a and Mmp3. * p<0.05, ** p<0.01,*** p<0.001. (C) Expressionof AC006064.4-201 in HCs after cheating with different concentrations ofDoxorubicin .  * p<0.05. Supplementary Figure 2. (A) QRT-PCR ofAC006064.4-201 in HCs when AC006064.4-201 was knocked down.  *** p<0.001. (B) QRT-PCR ofAC006064.4-201 in HCs when AC006064.4-201 was overexpressed.  ** p<0.01. (C) Western blot ofYBX3, APOBEC3C, PTBP1, LIN28A and IMPDH1 when they were separately knockeddown. (D) Knock down efficiency of CDKN1B shRNAs assessed by qRT-PCR.  *** p<0.001. (E) MRNA levels ofMmp3, Mmp13, Sox9, Aggrecan, p16INK4a, p21 and p53 assessed by qRT-PCR whenCDKN1B was knocked down in HCs.  * p<0.05,** p<0.01, *** p<0.001. (F) QRT-PCR of CDKN1Bin HCs when CDKN1B was overexpressed.  ** p<0.01.(G) MRNA levels of Mmp3, Mmp13, Sox9, Aggrecan, p16INK4a, p21 and p53assessed by qRT-PCR when AC006064.4-201 was overexpressed or co-overexpressedwith CDKN1B.  * p<0.05, ** p<0.01, *** p<0.001. Supplementary Figure 3. (A) Sequence ofAC006064.4-201.  (B) Knock down efficiency of Gm49317-201 ASOs assessed by qRT-PCR.  ** p<0.01. (C) Western blot ofPTPB1 in MCs aftering pulled-down with the biotinylated Gm49317-201 mixed probes. (D) Knock down efficiency of CDKN1B shRNAs assessed by qRT-PCR.  *** p<0.001. Supplementary Figure 4. (A) Representative images and flurence intensities of IF staining forCDKN1B and Col2a1 in mice cartilage of different groups. (n=10) * p<0.05, ** p<0.01, *** p<0.001. Scale bars, 200µm and 50µm. Supplementary Figure 5. (A) Representative images and flurence intensities of IF staining forCDKN1B and Col2a1 in mice cartilage of different groups. (n=10) * p<0.05, ** p<0.01. Scale bars, 200µm and 50µm.Additional file 2: Supplementary Table 1. RNAseq analysis. Supplementary Table 2. RPD-MS analysis. Supplementary Table 3. Primer sequences used in this study. Supplementary Table 4. Sequences of ASOs, shRNAs and probes. Supplementary Table 5. Antibodies usedin this study.


Background: The Japanese drug use system allowed the once-daily use of inhaled corticosteroid fluticasone furoate (FF) combined with a long-acting beta-2 agonist vilanterol (VI) and a long-acting muscarinic antagonist umeclidinium (UMEC) against asthma on 18 February 2021. We investigated the real-world effects of these drugs (FF/UMEC/VI) mainly on lung function tests.Methods: This was an open-label, uncontrolled, within-group time-series (before-after) study. Prior asthma treatment  was switched to FF/UMEC/VI 200/62.5/25 μg. Subjects were evaluated by lung function tests prior to, and 1–2 months after, initiation of FF/UMEC/VI 200/62.5/25 μg. Patients were asked questions regarding the asthma control test and preference for drugs.Results: Overall, 114 asthma outpatients (97% Japanese) were enrolled from February 2021 to April 2022: 104 subjects completed the study. Forced expiratory volume in 1 s, peak flow, and asthma control test score of FF/UMEC/VI 200/62.5/25 μg-treated subjects were significantly increased . In contrast with FF/VI 200/25 μg, instantaneous flow at 25% of the forced vital capacity and expiratory reserve volume were significantly increased by FF/UMEC/VI 200/62.5/25 μg . Sixty-six percent of subjects declared they wanted to continue FF/UMEC/VI 200/62.5/25 μg in the future. Adverse effects, mainly local, were seen in 30% of patients, but no serious adverse effects were seen.Conclusion: Once-daily FF/UMEC/VI 200/62.5/25 μg was effective against asthma without serious adverse events. This is the first report that demonstrated FF/UMEC/VI dilated peripheral airways using lung function tests. This evidence on drug effects may improve our understanding of pulmonary physiology and the pathophysiology of asthma. Asthma is defined by a history of respiratory symptoms including wheezing, shortness of breath, dyspnea, and cough that vary over time and intensity, together with variable expiratory airflow limitation . This co2-agonist (LABA) and ICS was recommended for patients at high risk of asthma exacerbations  overlap, termed ACO . The Japreceptor . UMEC wareceptor . Historireceptor .1), the efficacy of this combined drug (FF/UMEC/VI 200/62.5/25 μg) on other parameters of lung function tests has not been well known (2-agonists in asthma patients (The Japanese drug use system first approved the use of the triple combined drug consisting of ICS (FF 200 μg)/LABA (VI 25 μg)/LAMA (UMEC 62.5 μg) against asthma on 18 February 2021. Although there was a landmark report of the phase 3A random controlled trial (RCT) (CAPTAIN Study) on the data of forced expiratory volume in 1 s  study.We recruited asthma outpatients at the IUHW Shioya Hospital  from February 2021 to April 2022 . Recruitment was conducted during the outpatient service by physicians of this Hospital. All subjects provided written consent before participating in this study. Registration site and number: UMIN000047506.Inclusion criteria included: age ≥20 years, stable asthma with the use of ICS alone or ICS/LABA or ICS/LABA/LAMA, and unstable asthma with or without the use of inhalation therapy. Patients had to be diagnosed with moderate or severe asthma by pulmonary physicians .Exclusion criteria were pregnancy, unstable asthma that needed an increase in systemic steroids, a recent history of life-threatening asthma, and/or concomitant life-threatening disease.Previous inhalation treatments against asthma were switched to the once-daily use of FF/UMEC/VI 200/62.5/25 μg. For asthma patients without the use of inhalation treatments, FF/UMEC/VI 200/62.5/25 μg was initiated. Subjects were evaluated by lung function tests prior to, and 1–2 months after the initiation of FF/UMEC/VI 200/62.5/25 μg. The interval was shortened from 2–3 months to 1–2 months compared with our previous study of FF/VI 200/25 μg to reduce any bias caused by “lost to follow-up” . CHESTACt-test was used for comparisons between baseline and after the use of FF/UMEC/VI 200/62.5/25 μg (two-tailed). Statistical significance was set at p < 0.05. For the statistical analyses, Ekuseru-Toukei 2010  was used. The sample size was estimated according to our previous study on FF/VI 200/25 μg (n = 107)  or too late for after data  as missing lung function test data and thus excluded these patients’ data from the analysis.Data are shown as the mean ± standard deviation (SD). The Student’s paired n = 107) . To compwas 0.44 . To achit-test) conducted at different time points, and it was considered appropriate to compare the results of each before-after study in parallel and observe how significant differences emerged  were enrolled from February 2021 to April 2022. Most patients were satisfied with their current treatment with regular maintenance inhalation therapy with ICS alone or ICS/LABA or ICS/LABA/LAMA. In two uncontrolled asthma patients, inhalation therapy was not used before the use of FF/UMEC/VI 200/62.5/25 μg. Patient characteristics  at baseline included: age 71.8 ± 11.8/65.4 ± 18.6 years, height 157 ± 10/159 ± 9 cm, weight 61.8 ± 14.7/61.4 ± 15.4 kg, body mass index 25.0 ± 5.1/24.0 ± 4.9 kg/mPrevious inhalation drugs used before the use of FF/UMEC/VI 200/62.5/25 μg are shown in p < 0.001), V75 (p < 0.001), V50 (p < 0.001), V25 (p < 0.01), V25 to height (p < 0.01) and maximum mid-expiratory flow rate (p < 0.001) were significantly increased after the (switching) use of FF/UMEC/VI 200/62.5/25 μg. The p-value of V50 was the smallest (0.00014) among these airflow data. The p-value before (1st line) and after (2nd line) the omission was consistently almost the same. Most of the statistical results  were similar between this study and our previous study using FF/VI 200/25 μg, except for V25, V50/V25, and V25/HT (Changes in various airflow data are shown in the first lines of d V25/HT .p < 0.01), FEV1 (p < 0.001), percent predicted FEV1 (p < 0.001), vital capacity (VC) (p < 0.05), percent predicted VC (p < 0.05), forced vital capacity (FVC) (p < 0.05), percent predicted FVC (%FVC) (p < 0.01), and expiratory reserve volume (ERV) (p < 0.05) were significantly increased after the (switching) use of FF/UMEC/VI 200/62.5/25 μg in asthma patients. The p-value of FEV1 was extremely low (0.000050). The p-values on VC, percent predicted VC, FVC, %FVC, and ERV after the omission were a little larger than the p-values before the omission. Most of the statistical results  were similar between this study and our previous study using FF/VI 200/25 μg, except for FVC, %FVC, ERV, and IRV  was significantly increased only by FF/VI 200/25 μg. These findings were consistently seen regardless of the data omission.Comparisons between the impact of the rollout of FF/UMEC/VI 200/62.5/25 μg and FF/VI 200/25 μg on various airflow data from asthma patients are shown in By comparing the data before and after the (switching) use of FF/UMEC/VI 200/62.5/25 μg for asthma patients, we obtained the following findings. First, many lung function test data were significantly increased by the (switching) use of FF/UMEC/VI 200/62.5/25 μg. Among these data, significant increases in V25 and ERV were thought to be especially important. All data were compared with our previous study of FF/VI 200/25 μg . Second,The interval between the day of initiating the new inhalation drug and the day of obtaining the after data was shortened from 2–3 months to 1–2 months compared with our previous study on FF/VI 200/25 μg . The numFF/UMEC/VI 200/62.5/25 μg significantly increased the Peak flow, V75, V50, and V25. In contrast, FF/VI 200/25 μg significantly increased the Peak flow, V75, and V50, but did not significantly increase V25 . To the FF/UMEC/VI 200/62.5/25 μg did not significantly increase the IRV, but significantly increased the ERV and FVC. In contrast, FF/VI 200/25 μg significantly increased IRV, but did not significantly increase ERV or FVC. These findings were consistently seen regardless of the data omission of subjects who did not use inhalation drugs for more than 3 months before the initiation of FF/UMEC/VI 200/62.5/25 μg. The main targets of FF/VI 200/25 μg seemed to be relatively larger bronchi compared with FF/UMEC/VI 200/62.5/25 μg. Adding UMEC was thought to allow the expiration of more air and increase the FVC . To the LAMA drugs were first approved for COPD, then asthma . For exa3 receptors are expressed on smooth muscle cells and lung submucosal glands, and they regulate contraction, and mucus production and secretion  was thought to be mainly due to an old cerebral infarction. Therefore, there were no serious side effects related to this drug combination.1), but it did not lead to a significant reduction in moderate and/or severe exacerbations. Clinically meaningful changes, such as in acute exacerbation frequency, can be observed by longer use of drugs; therefore, RCTs with a longer observation period may be better than our before-after study. The CAPTAIN Study observed asthmatic patients for 24 weeks and reported the efficacy and safety of FF/UMEC/VI 200/62.5/25 μg. However, data related to V25, ERV, and IRV were not reported in the RCT. The strength of our study is that we observed the precise changes in lung function and that all participants received FF/UMEC/VI 200/62.5/25 μg. It was evident that the majority (66%) of subjects preferred FF/UMEC/VI 200/62.5/25 μg to previous drugs. On the other hand, 93% of subjects preferred FF/VI 200/25 μg to previous drugs in our previous study  of the use of a new drug is more realistic. This is an additional strength of our study.t-test) conducted at different time points, and it was considered appropriate to compare the results of each before-after study in parallel and observe how significant differences emerged (Although multiple regression analysis, Wilcoxon signed rank test, and Bonferroni multiple comparisons were considered, there was no appropriate method for rigorously comparing before-after studies (paired  emerged . This meIn conclusion, FF/UMEC/VI 200/62.5/25 μg was effective against asthma without any serious adverse events. Adding UMEC 62.5 μg to FF/VI 200/25 μg was thought to dilate the peripheral and very small airways additionally or synergistically. This is the first report that demonstrated FF/UMEC/VI 200/62.5/25 μg significantly increased V25 and ERV in asthma patients. Therefore, this is the first report that demonstrated FF/UMEC/VI 200/62.5/25 μg dilated peripheral and very small airways using lung function tests. This evidence on drug effects may improve our understanding of pulmonary physiology and the pathophysiology of asthma.


In vitro antibacterial screening revealed that several derivatives exhibited remarkable growth inhibition against different targeted microorganisms. All tested compounds showed excellent activity against the Micrococccus luteus strain (93.75 µg/mL ≤ MIC ≤ 375 µg/mL) as compared to the reference drug tetracycline (MIC = 500 µg/mL). Compound 4e bearing a p-chlorophenyl group on the pyrrolidine ring exhibited the greatest antifungal potential toward Candida albicans and Candida krusei  as compared to Amphotericin B . The target compounds were also tested in vitro against the lipoxygenase-5 (LOX-5) enzyme. Compounds 4i and 4l showed significant inhibitory activity with IC50 = 1.09 mg/mL and IC50 = 1.01 mg/mL, respectively, more potent than the parent drug, diclofenac sodium (IC50 = 1.19 mg/mL). In addition, in vivo evaluation of anti-inflammatory and analgesic activity of these spirooxindoles were assessed through carrageenan-induced paw edema and acetic acid-induced writhing assays, respectively, revealing promising results. In silico molecular docking and predictive ADMET studies for the more active spirocompounds were also carried out. In a sustained search for novel potential drug candidates with multispectrum therapeutic application, a series of novel spirooxindoles was designed and synthesized via regioselective three-component reaction between isatin derivatives, 2-phenylglycine and diverse arylidene-imidazolidine-2,4-diones (Hydantoins). The suggested stereochemistry was ascertained by an X-ray diffraction study and NMR spectroscopy. The resulting tetracyclic heterocycles were screened for their in vitro  Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs in clinical practice to relieve arthritis pain and inflammation ,2. They Pseudomonas aeroginosa and Staphylococcus aureus are among the leading factor of infections and increased healthcare costs -2,2″,5-triones 4 in 76−91% isolated yields  corresponding to H-4 proton of the pyrrolidine ring which showed 1H-13C HSQC correlation (J = 9.6 Hz and J = 4.8 Hz), attributed to the pyrrolidine proton H-5. The multiplicity and the coupling constant values of these protons confirm the formation of the regioisomer 4i as in the case of hypothetical regioisomer 4i′    with the4i, whose molecular structure is depicted in (i) carbonyl group of hydantoin part  are in trans-relationship, and (ii) a cis -relationship with H-5 pyrrolidinyl proton bonded to C-5 . The regio- and stereochemistry of the 1,3-dipolar cycloaddition was further corroborated by a single-crystal X-ray analysis of spiropyrrolidine 4 are formed through an endo-approach between the (Z)-5-arylideneimidazolidine-2,4-diones 3 and the in situ generated -dipole 1d, as outlined above in Accordingly, we propose that spirooxindole-hydantoins 4i shown in 2O molecules. The stereochemistry of the tetracyclic core bearing two additional aryl groups at C19 and C12 is in the solid state in line with the NMR data in solution and establishes the transoid arrangement of two the hydrogen atoms of the pyrrolidine-type five-membered cycle. As visualized in d(N1-H1···O6) 1.96(2) Å), the N1–H1···O6 angle being 173(2)°. Furthermore, a second intermolecular hydrogen bond is formed between N3–H3 and O4 (d(N5–H5···O5) 2.05(2) Å), The thus generated supramolecular 1D ribbon displays a third somewhat weaker N–H···O=C bonding between N7–H7 and O1 (d(N7–H7···O1) 2.13(2) Å) with a N7–H7···O1 angle being 169.4(19)° as well as a forth interaction between N6–H6 and O23 (d(N6–H6···O2) 2.11(2) Å). In addition, the co-crystalized H2O molecule interconnects two ribbons through O–H···O bonding with O3 (d(O7–H7B···O3) 1.87(3) Å) and N–H···O3 bonding (d(N2–H2···O7) 1.88(2) Å). Finally, a weak intermolecular C–H···F1 contact (d(C2–H15···F2) 2.49 Å) completes the supramolecular interactions occurring in this highly organized network   to produce hydroperoxides ,45. Frac50 values (concentration causing 50% enzyme inhibition) of the tested compounds and the reference drug diclofenac sodium are recorded in To evaluate the prospects of the newly synthesized spirooxindoles as promising drug candidates, we first studied their inhibitory potency against the Lipoxygenase-5 (LOX-5) enzyme. The IC50 = 1.01–4.96 mg/mL) against LOX-5 enzyme. Interestingly, spirooxindoles 4i and 4l  showed improved LOX-5 inhibition profile better that of diclofenac sodium (IC50 = 1.19 mg/mL) (As shown in 9 mg/mL) .4f (IC50 = 4.92 mg/mL) and 4g (IC50 = 4.96 mg/mL) bearing the N-benzyl substituent, were comparably less active than the other tested heterocycles. This result could be explained by means of the steric effect of the benzyl group resisted the binding ability with the LOX-5 enzyme. The introduction of halogen substituents  or hydroxyl group (4l–m) at the para position of the aromatic ring of the hydantoin, caused a remarkably increase of the in vitro LOX-5 inhibitory activity. This could be due to the lipophilicity of these groups improving the bioavailability. In addition, compound 4i and 4l featuring an unsubstituted isatin ring expressed the highest inhibitory potency. A brief analysis of the SAR of the newly spirooxindole-hydantoin derivatives revealed the following: compounds 4i and 4l, which showed the more potent in vitro lipoxygenase inhibitory, were accessed for their in vivo anti-inflammatory activity using carrageenan-induced rat paw edema method as reported by Lopes et al. [https://en.wikipedia.org/wiki/Carrageenan (accessed on 16 October 2023). Results recorded in 4i and 4l reduced the edema volume by 33% and 32%, respectively, 4 h after the administration of these compounds.Compounds s et al. . Diclofe4i and 4l was assessed by the acetic acid-induced writhing test using diclofenac sodium as positive controls as reported previously [The in vivo analgesic activity of the candidate drugs eviously ,50,51,52Acetic acid injection induces peritoneal inflammation in mice which reacts with a characteristic stretching behavior, called writhing. Any writhing was considered a positive response. The efficacy and potency of analgesic activity of the test compounds is inferred from the reduction in the frequency of writhings .4i and 4l to acetic acid-induced pain in rats exert good analgesic activity. In fact, the administration by injection of acetic acid to control rats provoked 63 instances of writhing .As can be seen in writhing . Pre-tre4i and 4l up to a dose of 16.25 mg/kg to rats did not cause any toxicity or physiological or biochemicals changes in the liver (AST and ALT activities) and –kidneys  functions. The acute toxicity test  showed t4a–m were selected to be screening for their antimicrobial activities, using in vitro micro diffusion method, against multiple bacterial strains , and two pathogenic reference yeasts namely Candida albicans ATCC 90028 and Candida kreusei ATCC 6258. In this part, the spiropyrrolidines 4a–m exhibited excellent antibacterial activity against M. luteus strain with MIC values ranging from 93.75 to 375 µg/mL. The spiropyrrolidines 4c, 4h, 4g, and 4m were the most potent derivatives with MIC values of 93.75 µg/mL, as compared to the reference antibiotic Tetracycline  (S. aureus pathogen was obtained for compound 4g (MIC = 23.34 μg/mL) followed by 4b, 4e, and 4l with MIC value of 46.87 µg/mL. We also noticed a moderate growth inhibition of the tested compounds towards E. coli (187.50 μg/mL ≤ MIC ≤ 750 μg/mL) and P. Aeroginosa (93.75 μg/mL ≤ MIC ≤ 187.50 μg/mL). Notably, compounds 4c and 4m exhibited significant antibacterial activity towards E. coli (187.50 µg/mL in MIC), higher than that of Tetracycline (MIC = 256 μg/mL). It is worth noting that compound 4e demonstrated strong antifungal activity against both the antifungal strains C. albicans and C. krusei with MIC values of 23.43 and 46.87 µg/mL, in comparison with Amphotericin B .The results showed that all synthesized compounds 0 µg/mL) . The besTo further determine the killing efficiency of the synthesized spiropyrrolidines against selected strains, the minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC) were assessed by the agar method . The resE. coli, and compounds 4b, 4d, 4f, 4g, 4h, and 4m revealed better efficacy  than the standard tetracycline reference drug (MBC of 512 µg/mL). Interestingly, most of the synthesized molecules exhibited good MBCs results versus Gram-negative pathogen Tetracycline was used as reference drug for the antibacterial assay while Amphotericin B was used as a standard antifungal drug. The results are summarized in the histograms presented in 4b and 4c were effective against the bacterial strain P. aeruginosa, while 4h and 4m were active against M. luteus (MBC = 187.50 µg/mL). However, no significant antibacterial activity was observed towards the Gram-positive bacteria S. aureus. The MFC results of the tested fungal strains showed that C. albicans is more sensitive to all spiropyrrolidines (except for 4d and 4h). The MFC values ranged from 23.43 to 187.50 μg/mL, while for Amphotericin B, it was recorded as 500 μg/mL. Compound 4e also showed a good killing efficacy against the C. krusei strain, with an MBC value of 46.87 μg/mL. Notably, compounds 4a–m on selected organisms was determined using their MBC/MIC ratio. The compounds are considered as bactericidal or fungicidal agents when the MBC/MIC or MFC/MIC ratios ≤ 4 and as bacteriostatic or fungistatic if these ratios are >4. All the tested spiropyrrolidines exhibited a bactericidal effect (MBC/MIC ≤ 4) against gram positive and Gram-negative bacteria, except derivatives 4c, 4g, and 4h that demonstrated a bacteriostatic effect (MBC/MIC = 8 > 4) against M. luteus pathogen and 4b showing a bacteriostatic action towards S. aureus strain (MBC/MIC = 16). For the fungal strains, the majority of the synthesized heterocycles can be classified as fungicidal agents, especially compounds 4a, 4b, 4e, 4g, 4i, 4k, 4l, and 4m which showed MFC/MIC ratios ≤ 4 on both strains C. Albicans and C. Krusei.The bactericidal and bacteriostatic action of the spiropyrrolidines 4b and 4d with electron-donating groups (CH3 and OCH3) at the para-position of phenyl ring exhibit better inhibition against S. aureus than derivatives 4e and 4h containing chloro substituent attached to the same phenyl ring. Moreover, compounds with electron-donor substituents attached to the phenyl ring of the dipolarophile part generally displayed a higher inhibitory potency than derivatives incorporating electron-withdrawing groups bonded to the identical phenyl ring.The SAR was established to unravel the important features responsible for the antimicrobial activity of the tested compounds. The influence of different substituents on the isatin nucleus as well as on the aromatic cycle of the dipolarophile was therefore explored. Biological assays results indicated that compounds 3, OCH3 and OH) at the para and ortho positions of the phenyl ring are more effective against Gram-negative bacteria P. aeroginosa. For instance, spiropyrrolidines 4b, 4c, and 4d feature electron-donating substituents (CH3 and OCH3) at the para-position of the phenyl ring exhibited better inhibition compared to compounds containing halogen groups  attached to the same phenyl ring.Derivatives that had an electron-donating groups . Notably, the inhibitory activity is linked to both the substitutions on the dipolarophile and isatin core.For the Gram-negative strain M. luteus compared to tetracycline, regardless of the nature of the substituents present on both the isatin and the dipolarophiles unit. However, a noteworthy observation is that the activity generally increases by an electron-withdrawing group attached to the isatin core and decreases in the case of unsubstituted isatin- derivatives . All compounds are more active against 4c, 4g, and 4h were docked into the active site of three bacterial targets: Thymidylate Kinase (TMK) (PDB ID 4QGG) from S. aureus, (3-oxoacyl-[acyl-carrier-protein) synthase 3 (PDB ID 4EWP) from M. luteus, DNA gyrase (PDB ID 4DUH) from E. coli. The ligand 4e was docked to the target secreted aspartic protease (PDB ID 3Q70) from C. albicans and C. krusei. The Ligands 4i and 4l with the most promising ant-inflammatory potential were docked into the active sites of LOX-5 (PDB ID: 3V99). The PDB binding structures of different target enzymes are presented in In an attempt to rationalize the observed bioactivity for the newly synthesized derivatives, molecular docking studies were performed. The most antibacterial spirooxindole-hydantoins, i.e., 4c, 4g, 4h, 4e, 4i and 4l were measured computationally in terms of their MolDock Score. MolDock Score is actually the strength of the intra-molecular interactions among multiple molecules within the binding pockets of selected targets. After having the molecular docking calculation for all 4c, 4g, 4h, 4e, 4i and 4l compounds with their selected enzymes, the results obtained were analyzed based on the different parameters such as: Affinity (S-score), Interactions (types and distances). First, if an E–L formed complex gives a low energy score it means that this complex is the most stable also we have high affinity between the compound and target. Second, hydrogen bonds are the most important in the formation of E–L complexes and they can be classified according to the following intervals: If the distance belongs to the interval between 2.5 Å and 3.1 Å, we are considered to be strong interactions. If the distance belongs to the interval between 3.1 Å and 3.55 Å, we are assumed to be weak [.The potencies of the most bioactive compounds:  be weak ,57. Thir be weak  who sugg be weak ,60.4c, 4g, and 4h with the bacterial targets have been grouped in The results obtained after the docking calculations and the best pose received for the compounds 4g is the most potent antibacterial agent against TMK (ID: 4QGG) from S. aurues, revealing the highest negative score of −153.700 kcal/mol, comparable to the standard drug Tetracycline with the score of −112.465 kcal/mol  from S. aurues which showed that was formed almost the same number of interactions with the TMK protein of S. aureus compared to the Tetracycline with a small difference in the type of residue  with crucial residues: ARG36 (1.74 Å), GLU37 (2.26 Å), PRO38 (2.12 Å) and ARG48 (2.73 and 3.05 Å) of TMK protein from S. aureus (4g displayed two hydrophobic interactions (Pi–Pi T-shaped) with the residues PHE66 and TYR100. In addition, two other hydrophobic interactions (Pi-Pi Stacked) were formed between compound 4g and residue PHE66. While four hydrophobic interactions (Pi-Alkyl) with active site residues: ARG36, ILE47, ARG48 and VAL51  for molecular docking analysis. After solving that problem, we launched molecular docking simulation and as presented in 4h fits well in the binding pocket of M. luteus receptor (PDB ID 4EWP) with highest negative score of −147.875 kcal/mol, which is comparable of Tetracycline with a score of −96.245 kcal/mol. Moreover, the docking conformation of compound 4h showed that this compound established three hydrogen bonds and five hydrophobic interactions with active site residues of the target protein (4h to the (3-oxoacyl-[acyl-carrier-protein) synthase enzyme vs. the Tetracycline. Furthermore, previous papers [Due to the existence of only one crystal structure of the  protein . The hyd protein . While f protein . On the s papers ,65 confi4c binds into the DNA Gyrease enzyme. The MolDock Score energy and types of possible interactions in addition to, the interacting moiety of our docked compound is represented in The obtained results revealed that the compound 4c proved a good ability to the binding into DNA Gyrase binding site and this is confirmed by forming 6 strong hydrogen bonding interactions with Gly 101(1.43 Å), Arg 136(2.63 Å and 2.23 Å), Gly 77(2.26 Å), and Lys 103(1.86 Å). As shown from the obtained data compound, 4c was able to form 6 hydrophobic interactions with different amino acids in the DNA Gyrase binding pocket which are ARG76, LYS103, ILE94, PRO79, ILE78, and GLY77. In addition, the compound 4c showed two electrostatic interactions (Pi-Anion and Pi-Cation) with GLU50 and LYS103, as displayed by orange dotted lines in 4c  . Also, mkcal/mol . However studies ,67,68.4e with the fungal target are given in The results obtained after the docking simulations and the best pose received for the compound 4e being the most potent antifungal agent displaying a good docking MolDock Score of −129.886 kcal/mol and many molecular interactions with secreted aspartic protease (SAPs) enzyme from C. Albicans vs. of the standard drug Amphotericin B with the score of −112.504 kcal/mol. Also, we note that this compound fits well in the binding pocket of the secreted aspartic protease (SAPs) , which showed good interactions. In fact, compound 4e established four strong hydrogen bonds, two hydrophobic interactions and one electrostatic interaction with active site residues of the target protein , THR222(2.02 Å) and THR221 of the target protein, the secreted aspartic protease (SAPs) (4e with the secreted aspartic protease (SAPs) vs. the Amphotericin B, which are confirmed in previous research [The hydrogen bonds were observed between the compound e (SAPs) . While te (SAPs) . Neverthresearch ,70.4i, 4l and Diclofinac sodium was carried out -NH/bond distance = 2.79 Å and H/ASN554(A)-OD1/bond distance = 2.47 Å, another one carbon H-bonds H/ASN554(A)-OD1/bond distance = 2.54 Å (A7 and ASN554(A). During which, this compound formed binding with the Fe2+ ion of the enzyme’s active site.Molecular docking of the compounds ried out . Regardiectively . Additioc sodium . The doc= 2.54 Å . A halogπ-alkyl interactions) with the following residues: ILE406 (A), ALA410 (A), LEU607 (A), VAL175 (A) and ALA672(A) -HE22/bond length = 1.67 Å, H/VAL175(A)-O/bond length = 1.91 Å and H/ASN554(A)-OD1/bond length = 2.23 Å. Additionally, another two carbon H-bonds, O/HIS367(A)-HE1/bond distance = 2.33 Å, and H/ASN554(A)-OD1/bond length = 2.57 Å. This compound indicates direct binding with the Fe2+ ion of the enzyme’s active site, and a metal–ligand interaction was observed in this case. Finally, compound 4l exhibits four hydrophobic interactions  with the following residues: ILE406(A), ALA410(A), LEU607(A), LEU607(A) and ALA672(A)  a. In thiLA672(A) b. These http://www.swissadme.ch/, accessed on 20 May 2023) and the pkCSM server , and all results are given in ADME-T and drug-likeness parameters were calculated using both online servers SwissADME server  is: <5 (n-HD: (0~7)) and the number of hydrogen bond acceptors is <10 (n-HA: (0~10). Furthermore, all these compounds have molecular weights ranging from 100 to 500 g/mol except compounds 4g and 4h, and MLogP and WLogP values are <5. In addition, we found nROTB values <11, indicating that these compounds are flexible. Also, all TPSA values obtained were below 140 Å. Finally, these results show that all compounds satisfy all criteria of drug-likeness without presenting any violation of the Lipinski, Veber and Egan rules. Evidently, no problems are caused in oral bioavailability and pharmacokinetic parameters of either compound.It can be seen from 4c, 4g, 4h, 4e, 4i and 4l) have good Caco-2 permeability . Furthermore, it was evidenced that all compounds had HIA values greater than 30%, indicating that these compounds were administered orally, and that they are strongly absorbed by the gastrointestinal system into the bloodstream of the human body.In addition, the analysis in 4g and 4h are >−2 which means that these compounds are able to penetrate the CNS. On the other hand, we note that the other compounds  are not able because of their weak logPS values , this indicates that both compounds are poorly distributed to the brain, comparable to other compounds  which are poorly distributed.According to data from this table, we can see that logPS values for both compounds S values . AdditioData from this table indicate that all compounds are inhibitors of CYP2C19 isoform and CYP3A4 substrate. Furthermore, neither compound is a CYP1A2 inhibitor nor is it a CYP2D6 substrate . Further1H NMR, 101 MHz for 13C {1H} NMR) spectra were recorded on Bruker Avance 400 machine . The chemical shifts δ were reported in ppm relative to tetramethylsilane (TMS). Data were described as follows: chemical shift (δ in ppm), multiplicity , coupling constants (Hz), integration. Optical rotations were determined by a Perkin Elmer polarimeter. Elemental analyses were performed on a Perkin Elmer 2400 Series II Elemental CHNS analyzer . Thin-layer chromatography (TLC) was performed on silica gel plates   using UV light at 254 nm.The NMR (400 MHz for 3 (1 mmol), 2-phenylglycine 2 (1 mmol) 2, and isatin 1 (1 mmol), in methanol (5 mL) was heated under reflux for 5 h. The solvent was subsequently removed under vacuum. The residue was chromatographed on silica gel employing ethylacetate-cyclohexane (4:6 v/v) as eluent, with the addition of few drops of methanol to obtain the pure products 4.A mixture of 3-arylideneimidazolidine-2,4-dione iso (H) = −1.2 Ueq(C) for CH2 and CH and with Uiso(H) = −1.5 Ueq(C) for CH3. All H atoms were refined freely using independent values for each Uiso(H).Data collection was performed on a Bruker D8 Venture four-circle diffractometer from Bruker AXS GmbH . CPAD detectors used were Photon II from Bruker AXS GmbH; X-ray sources: Microfocus source IμS; and microfocus source IμS Mo and Cu, respectively, from Incoatec GmbH with mirror opticsHELIOS and a single-hole collimator from Bruker AXS GmbH. Programs used for data collection were APEX4 Suite   Å, b = 12.5291(10) Å, c = 15.0421(11) Å, α = 95.793(3)°, β = 91.329(3)°, γ = 99.674(3); V = 2236.9(3) Å3, Z = 4, Dcalc = 1.341 g/cm3, T = 100 K, R1 = 0.0509, wR2 = 0.1291 for 59257 reflections with I ≥ 2σ (I) and 14895 independent reflections. GOF = 1.052. Largest diff. peak/hole/e Å−3 0.61/−0.33. Data were collected using graphite monochromated MoKα radiation λ = 0.71073 Å and have been deposited at the Cambridge Crystallographic Data Centre as CCDC 2248684.  activity was determined by the protocol of by Choudhary et al. . Analges4c, 4g, 4h, 4e, 4i and 4l were pre-optimized by means of the Molecular Mechanics using Force Field MM+. After that, all compounds optimized using the semi-empirical method AM1 [The 3D structures of four most active compounds: thod AM1  which imthod AM1 .https://www.rcsb.org/, accessed on 20 May 2023), accessed on 10 May 2023 that is, Thymidylate Kinase (TMK) (PDB ID 4QGG) from S. aureus [M. luteus [E. coli [C. albicans and C. krusei [The X-ray crystallography structures of four different drug target proteins were downloaded from the RCSB Protein Databank   softwareRe-docking of the co-crystal ligands was performed to validate the applied molecular docking approach. We found that the root mean square deviation (RMSD) values variation are between 1 and 2 Å  for all http://www.swissadme.ch/ accessed on 20 May 2023) [http://biosig.unimelb.edu.au/pkcsm/prediction, accessed on 20 May 2023) [Validation of drug-likeness rules, namely Lipinski, Veber and Egan, were conducted by calculating different parameters of physicochemical properties , and number of hydrogen bond donors (nHD)), by using the SwissADME server (ay 2023) . On the ay 2023)  is used (i) compounds 4c, 4h, 4g, and 4m  were more potent than the reference antibiotic tetracycline , against M. luteus (ii) compounds 4c and 4m showed highest antibacterial activity with a MIC value of 187.50 µg/mL against E. coli, and (iii) heterocycle 4e showed the greatest antifungal potential toward C. albicans and C. krusei  as compared to amphotericin B . On the other hand, all tested spirooxindoles exhibited modest to potent inhibitory activity (IC50 = 1.01–4.96 mg/mL) against LOX-5 enzyme. Among them, spirooxindoles 4i and 4l  showed improved LOX-5 inhibition profile better that of diclofenac sodium (IC50 = 1.19 mg/mL). Moreover, 4i and 4i showed a good anti-inflammatory activity  which was comparable to the reference standard diclofenac sodium (inhibition: 46%) in carrageenan-induced rat paw edema assay. In addition, they displayed acceptable in vivo analgesic activity in acetic acid-induced writhing response  compared to reference drug (63%). Finally, molecular docking simulation and ADME-T prediction revealed that compounds 4g, 4h, 4c, and 4e have high affinities with the pocket of bacterial and fungal targets, also compounds 4i and 4l have high affinities with the binding site of LOX-5 target, which was confirmed by a lowvalue MolDock score and the appearance of several types of interactions which formed between these compounds and residues of the active site of the target selected. Moreover, ADMET predictions and physicochemical properties proved that all rules: Lipinski, Veber and Egan rules were compiled by all compounds tested. Thus, some of newly synthetized spirooxindoles could act as a platform for further evaluation.In summary, a novel series of spirooxindole-hydantoins was successfully synthesized as potent anti-inflammatory, analgesic, and antimicrobial agents. Antimicrobial screening revealed that