Bauhinia bauhinioides kallikrein inhibitor, rBbKIm, which has shown negative effects on prostate tumor cell lines DU145 and PC3. Crataeva tapia bark lectin, CrataBL, targets sulfated oligosaccharides in glycosylated proteins and has also demonstrated deleterious effects on prostate and glioblastoma tumor cells. However, neither rBbKIm nor its derived peptides affected the viability of SK-MEL-28, a melanoma cell line, while CrataBL decreased viability by over 60%. Two peptides, Pep. 26 (Ac-Q-N-S-S-L-K-V-V-P-L-NH2) and Pep. 27 (Ac-L-P-V-V-K-L-S-S-N-Q-NH2), were also tested. Pep. 27 suppressed cell migration and induced apoptosis when combined with vemurafenib, while Pep. 26 inhibited cell migration and reduced nitric oxide and the number of viable cells. Vemurafenib, a chemotherapy drug used to treat melanoma, was found to decrease the release of interleukin 8 and PDGF-AB/BB cytokines and potentiated the effects of proteins and peptides in reducing these cytokines. These findings suggest that protease inhibitors may be effective in blocking melanoma cells and highlight the potential of CrataBL and its derived peptides.Melanoma is difficult to treat with chemotherapy, prompting the need for new treatments. Protease inhibitors have emerged as promising candidates as tumor cell proteases promote metastasis. Researchers have developed a chimeric form of the  Lectins are a class of proteins that reversibly bind to carbohydrates other than enzymes or antibodies. They are found in most living organisms, such as viruses, bacteria, plants, and animals , and areCrataeva tapia tree, belonging to the Capparaceae family, found in northeastern Brazil, and purified according to the Araújo protocol , an2) was named Pep. 26, which was synthesized based on the CrataBL reactive site region and Pep. 27 (Ac-L-P-V-V-K-L-S-S-N-Q-NH2) was the inverse of Pep. 26 with D-amino acids, resulting in Pep. 26 and Pep. 27 enantiomers. They were synthesized by Watson Bio , and their homogeneity, purity, and concentration were evaluated using reversed-phase high-performance liquid chromatography.The CrataBL-derived peptide (Ac-Q-N-S-S-L-K-V-V-P-L-NH4/well) and incubated for 24 h. Increasing concentrations of rBbKIm  or CrataBL  in DMEM High medium with 10% (for SK-MEL-28 cells) and 15% (for HFF-1 cells) of FBS and in combination with Vemurafenib (2 μM) were used to treat cells for 24 h, 48 h and 72 h. After the period, 10 μL/well of Prestoblue was added, the plate was incubated for 10 min at 37 °C, and absorbance was measured at 560–590 nm. The experiment was performed in triplicate and was repeated at least three times.The inhibitory effect was assessed using the Prestoblue reagent  . Cells w4) were seeded in 96-well microplates , and incubated for 24 h. Different concentrations  of CrataBL and protein-derived peptide have been used in combination with chemotherapy to treat for different periods . Plates were incubated with BrdU reagent for 24 h at 37 °C, the medium was removed, and the denaturation solution was added for 30 min. The denaturing solution was discarded, and an anti-BrDU antibody  with a dilution of 1:1000 was added for 1 h and 30 min. The wells were washed several times and the substrate was added for chemiluminescence emission analyzed at 405 nm using a FlexStation Multi-Mode Microplate Reader spectrophotometer . This experiment was performed in triplicate and was repeated at least three times.Bromodeoxyuridine (BrdU) Kit  was used to detect the synthesis of new Deoxyribonucleic acid (DNA) sequences . SK-MEL-5/mL of SK-MEL-28 cells were seeded and cultured at 37 °C, 5% CO2 for 24 h. After reaching 90–100% confluence, 10 µg/mL of mitomycin  was added 15 min before scraping. Using a P200, a risk was created in the center of the well, and a picture of the initial moment was taken (t = 0 h) with a camera (Sony Cyber-Shot) coupled to an inverted optical microscope. The medium was removed and the cells were treated with the respective drugs: 12.5, 25, 50, and 100 µM of CrataBL; 50, 100, and 200 µM pep. 26; 50, 100, and 200 µM pep. 27; and 2 µM of vemurafenib in each treatment. After 24 h, new photos were taken, as well as after 48 h. The size of the gap was quantified using the Image J program (ij153-win-java8), and graphs were plotted using GraphPad Prism 7 program [In 24-well plates, 1 × 10 program . The exp4 cells/250 µL and 100 µM of each treatment associated with 2 µM of vemurafenib dissolved in culture medium without FBS were added to the top of the formed matrigel. Inside each well, 400 µL of medium containing 10% SBF was added and incubated for 24 and 48 h. Cells that remained inside the insert were removed with a cotton swab and cells that invaded located in the external region were fixed with 4% formaldehyde (v/v). After 30 min, the wells were washed with phosphate-buffered saline (PBS) and stained with 4′,6-diamidino-2-phenylindole  for 15 min under slow shaking. The membranes were washed with PBS and the inserts were photographed with a camera connected to an inverted optical microscope . The stained nuclei of the cells were counted using the ImageJ program (ij153-win-java8), and graphs were plotted using the GraphPad Prism 7 program [In 24-well plates, Boyden Transwell/ThinCertsTM chambers  were used. Matrigel at a 1:6 dilution (in medium without FBS) was added to each insert and allowed to polymerize. After this period, 5 × 10 program .6 cells/well and treatment compounds  resuspended in DMEM High medium containing 10% FBS were added to the wells. After 4 h of incubation at 37 °C, the supernatant was removed, carefully so as not to touch the bottom of the wells, washed with PBS, and fixed with 4% paraformaldehyde for 1 h at 4 °C. Subsequently, the fixative was removed and the wells were washed again with PBS and stained overnight with toluidine blue. Excess dye was removed and the wells were washed with PBS. One hundred microliters of 1% sodium dodecyl sulfate (SDS) was added, and after 30 min, the fluorescence was measured at 540 nm using a Spectra Max Plus 384 spectrophotometer .Adhesion molecules such as collagen I (8 µg/well), collagen IV (4 µg/well), laminin (4 µg/well), vitronectin (0.2 µg/well), and fibronectin (4 µg/well) were added  in 96-well plates and placed at 4 °C for polymerization for 24 h. After this time, 100 µL/well of 1% bovine serum albumin (BSA) dissolved in PBS was added as a control substrate and incubated for 1 h at 37 °C. BSA was removed and plates were washed with PBS and 5 × 104 cells/well at 37 °C, 5% CO2 for 24 h. Subsequently, the culture medium was removed and cells were treated for 24 and 48 h with these compounds: 12.5, 25, 50, and 100 µM of CrataBL; 50, 100, and 200 µM pep. 26; 50, 100, and 200 µM pep. 27; and/or 2 µM vemurafenib for each treatment. After the treatment period, the supernatant was collected, the wells were washed with PBS, the cells detached with versene for 5 min, then the supplemented medium was added, and the Eppendorf tubes were centrifuged at 3500 rpm, for 5 min, at 25 °C. Cells were resuspended in a binding buffer containing 1.25 µL Annexin V/FITC (cellular fluorescein isothiocyanate) (30 µg/mL) and 1.25 µL Propidium Iodide (50 µg/mL) using the BD Pharmigen Kit . The incubation period was 10 min and readings were performed using a flow cytometer , with an analysis of at least 10,000 events [SK-MEL-28 cells were incubated in 24-well plates at a density of 5 × 100 events .6 cells) seeded in 6-well plates and incubated for 24 h. The culture medium was removed and concentrations of 100 µM of CrataBL, 100 µM of Pep. 26, 100 µM of Pep. 27, and 2 µM of vemurafenib in each treatment were added and its effect was evaluated after 24 h. To quantify proteins using micro bicinchoninic acid (microBCA)  the supernatants were collected and PBS containing 0.25 mM of sodium orthovanadate was added to the wells and successive scrapings were performed to detach the cells. The samples were centrifuged and the supernatants were discarded, this step was repeated and the precipitates were resuspended in lysis buffer containing a cocktail of protease inhibitors  and phosphatases (0.25 mM sodium orthovanadate). The samples were disrupted using several freezing and heating steps. After centrifugation, supernatants were collected. Thirty micrograms of total protein were added to each well and the gels (15%) were run [v/v) Tween 20) was added for 2 h, then the membranes were washed with TBST (buffer 25 mM Tris + 192 mM glycine and 0.1% (v/v) Tween 20) and then incubated with a primary antibody solution containing anti-FAK, Src, ERK, phosphorylated FAK, phosphorylated Src, phosphorylated ERK, NF-kB and Bax antibodies  in with a dilution of 1:1000 in (5% BSA), 200 mM Tris buffer, pH 8, containing 0.15 M NaCl and 0.05% Tween 20, under gentle stirring at 4 °C overnight. The next day, the membranes were washed with TBST and a secondary antibody solution (rabbit IgG)  with a dilution of 1:1000 (in 1% BSA (w/v) + 0.025 M Tris + 0.192 M glycine, and 0.1% (v/v) Tween 20) was added for 2 h and washed with TBS (without Tween 20). The membranes were then visualized using the photo documenter Bio-Rad/Bio-Imaging Systems after the addition of SuperSignal reagents  [SK-MEL-28   in a rat4/mL SK-MEL-28 cells were seeded and cultured at 37 °C, 5% CO2 for 24 h; after confluence was reached, cells were treated with 50 µM and 100 µM of CrataBL; 100 µM pep. 26; 100 µM pep. 27, and 2 µM of vemurafenib in each treatment and incubated for another 24 h. Cellular secretome was collected (100 µL) in Eppendorf tubes and stored in a freezer at −80 °C for later use. Protein quantification of the samples was performed using a microBCA , and cytokine dosages were analyzed using Luminex 200 equipment , where the evaluated molecules were PDGF-AB/BB, IL6, and IL-8. Forty micrograms of total protein were used for this analysis [The cell culture method for this dosage was similar to that used to measure NO levels. In 24-well plates, 5 × 10analysis .4/mL SK-MEL-28 cells were plated in a 24-well plate for 24 h at 37 °C, 5% CO2. After this period, the cells were treated with 50 and 100 µM CrataBL, 100 µM pep. 26, 100 µM pep. 27, and 2 µM vemurafenib in each treatment and incubated for an additional 24 h. Cellular secretome was collected (100 µL) in Eppendorf tubes and immediately frozen at −80 °C for later use. The quantification of the samples was performed using microBCA  and 200 µg of total protein was used for NO dosages using the Nitric Oxide Analyzer (NOATM 208i-Sievers) in which chemiluminescence originates from the conversion of nitrite to nitric oxide [5 × 10ic oxide  which isp < 0.05, ** p < 0.005, and *** p < 0.0005.In the data analysis, the GraphPad Prism 7 program was used where the statistical differences were determined by the One-Way ANOVA method, the Tukey test (SigmaPlot 10.0), and the data with mean + standard deviation. Statistical differences were considered significant at *


We conducted a prospective multicenter phase II study to evaluate the efficacy and safety of CapeOX as a first-line therapy for AGC in older patients.Capecitabine plus oxaliplatin 2 on days 1–14) and oxaliplatin (130 mg/m2 on day 1) every 3 weeks. After the initial feasibility assessment, the dose was reduced considering toxicity . The primary endpoint was overall survival (OS).Chemotherapy-naive patients aged ≥ 70 years with AGC were eligible. Initial treatment comprised capecitabine , and time to treatment failure (TTF) were 12.9 (95% CI 11.6–14.8), 5.7 (95% CI 5.0–7.0), and 4.3 (95% CI 3.9–5.7) months, respectively, for all patients; 13.4 (95% CI 9.5–16.0), 5.8 (95% CI 4.1–7.8), and 5.3 (95% CI 3.5–7.2) months in the original-dose group; and 12.8 (95% CI 11.3–15.3), 5.7 (95% CI 4.4–7.0), and 4.1 (95% CI 3.7–5.7) months in the reduced-dose group. The most common grade 3/4 toxicities were neutropenia (17.9%), anemia (12.8%), and thrombocytopenia (12.8%) in the original-dose group and neutropenia (13.8%) and anorexia (12.3%) in the reduced-dose group.These findings demonstrate CapeOX's efficacy and safety in older AGC patients. Gastric cancer is the third leading cause of cancer‐related death and the fifth most common malignancy diagnosed worldwide . The staBased on the results of a previous randomized controlled trial, the sixth edition of the Japanese Gastric Cancer Treatment Guidelines defines a regimen combining a platinum-based agent and a fluorouracil-based agent, including S-1 plus oxaliplatin (SOX), capecitabine plus oxaliplatin (CapeOX), and FOLFOX, as an acceptable first-line chemotherapy regimen for patients with epidermal growth factor receptor 2 (HER2)-negative AGC –9. WhileCapecitabine is an oral fluoropyrimidine rationally designed to preferentially generate 5-fluorouracil (5-FU) in tumors and might be a safer option than S-1 in older patients . AlthougTherefore, this prospective multicenter phase II study (UMIN000022450/jRCTs051180126) aims to examine the efficacy and safety of CapeOX in Japanese older patients with AGC.3, absolute neutrophil count ≥ 1,500/mm3, platelet count ≥ 100,000/mm3, total bilirubin level ≤ 1.5 mg/dL, serum transaminase ≤ 100 IU/L, serum creatinine ≤ 1.5 mg/dL, and creatinine clearance ≥ 50 mL/min; evaluable lesion according to the Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1  on days 1–14 of each course) and oxaliplatin (130 mg/m2 intravenously on day 1 of each course) every 3 weeks. As described by Chin et al., safety analyses were performed upon completion of the initial two courses of chemotherapy for the first 20 patients; the IDMC considered the tolerability of chemotherapy from the initial safety data, according to the protocol [The original-dose CapeOX treatment comprised capecitabine  and oxaliplatin (100 mg/m2 intravenously on day 1 of each course) every 3 weeks. Additionally, prophylactic antiemetic therapy for oxaliplatin was administered. Treatment delays, suspensions, or dose modifications were performed according to the J-CLASSIC study [2 in the original-dose group and 85 mg/m2 in the reduced-dose group. Oxaliplatin and capecitabine were discontinued in the event of grade ≥ 4 non-hematological toxicity. If an oxaliplatin-related allergic reaction or peripheral neuropathy occurred, capecitabine could be continued as monotherapy.Five patients were unable to complete the initial two courses of chemotherapy due to toxicity, necessitating dose reduction (10/15) or suspension of the subsequent course (12/15). Considering these results, the IDMC recommended continuing the study with a reduced-dose CapeOX treatment comprising capecitabine . The secondary endpoints were progression-free survival (PFS), time to treatment failure (TTF), response rate, and adverse events. Tumors were assessed every 8 weeks until disease progression, and objective responses were evaluated according to RECIST version 1.1 . OS was . reported that the median OS time for CapeOX therapy was 13.5 months [Our trial was designed as a therapeutic exploratory study to compare our CapeOX therapy with historical reference data in the first-line treatment of older patients with AGC. However, at the time of the trial’s planning, historical reference data for older gastric cancer were scarce. Koizumi and Imamura reported the results of S-1 monotherapy in older patients with AGC, with a median OS of 10.4 months and 9.2 months, respectively . In addi5 months . Hence, The primary analysis was based on the full analysis set (FAS), comprising all enrolled patients excluding those deemed ineligible after enrollment. We used the Kaplan–Meier method to estimate survival curves and Greenwood’s formula to calculate 95% confidence intervals (CIs) for survival rates. Log-rank test was applied to compare survival curves. Statistical analyses were conducted using SAS version 9.4 .Patients were enrolled from September 2016 to June 2020. After enrolling the initial 39 patients treated with original-dose CapeOX , the protocol was revised to incorporate IDMC recommendations, and the subsequent 69 patients were treated with reduced-dose CapeOX (reduced-dose group). The last follow-up analysis was conducted in July 2022. Finally, 108 patients from 26 centers in Japan were enrolled in this study. However, one patient with HER2-positive cancer and three patients with study drug violations—administered generic drugs—were excluded. A total of 104 patients were included in the FAS . Treatment was discontinued due to adverse events, patient complaints, and conversion surgery in 4 (10.3%), 4 (10.3%), and 2 (5.1%) patients in the original-dose group and 5 (7.7%), 5 (7.7%), and 3 (4.6%) patients in the reduced-dose group, respectively. Adverse events delayed treatment in 29 patients (74.4%) in the original-dose group and 40 patients (61.5%) in the reduced-dose group, and reduced the oxaliplatin and capecitabine dose in 26 cases (66.7%) and 26 patients (66.7%) in the original-dose group and 24 patients (36.9%) and 29 patients (44.6%) in the reduced-dose group, respectively.The median number of CapeOX treatment cycles was 6.0 in both groups. The median actual dose intensities in the original-dose and reduced-dose groups were 5732.8 mg/mn = 5; partial response, n = 39; stable disease, n = 15; progressive disease, n = 12; and not evaluable, n = 2). The ORR was 55.6% (95% CI 35.3–74.5%) in the original-dose group (n = 27) and 63.0% (95% CI 47.6–76.8%) in the reduced-dose group (n = 46). The disease control rate in the FAS, original-dose group, and reduced-dose group was 80.8% (95% CI 69.9–89.1%), 70.4% (95% CI 49.8–86.2%), and 87.0% (95% CI 73.7–95.0%), respectively. A waterfall plot of the best overall response for each patient is shown in Fig. The overall response rate (ORR) of patients with target lesions was 60.3%  months, and the 1-year OS rate was 56.4%  of patients, including second-line chemotherapy in 69% , surgery in 5%, and radiotherapy in 1%. Third-line chemotherapy was administered to 54% of patients , surgery to 1%, and radiotherapy to 1% of patients. Fourth-line, fifth-line, and sixth-line chemotherapy were administered to 14%, 7%, and 4% of patients, respectively.Post-study treatment was performed in 75%  anti-tumor drug widely used in Japan due to its outpatient convenience and mild toxicity. Several meta-analyses have reported that S-1 and capecitabine could be used in mono- and combination therapy for AGC –27, and In our study, the median OS, PFS, and TTF among all patients were 12.9 (95% CI 11.6–14.8), 5.7 (95% CI 5.0–7.0), and 4.3 (95% CI 3.9–5.7) months, respectively, and the primary endpoint was met. In the reduced-dose group, the median OS, PFS, and TTF were 12.8 (95% CI 11.3–15.3), 5.7 (95% CI 4.4–7.0), and 4.1 (95% CI 3.7–5.7) months, respectively, and were relatively equivalent to those in the original-dose group. Moreover, the incidence of severe hematologic toxicity and elevated creatinine levels in the reduced-dose group was lower than in the original-dose group, suggesting that reduced-dose CapeOX is more suitable for older patients than original-dose CapeOX.P = 0.108) and concluded that platinum-based combination chemotherapy, as compared with capecitabine monotherapy, was associated with a survival benefit in older patients with AGC [2. Recently, Hall et al. reported the results of the GO2 trial, comparing three doses of CapeOX  in older and frail patients with advanced gastroesophageal cancer, and showed that reduced-dose chemotherapy provided good persistence of quality of life and low toxicity without significantly compromising cancer control [Several studies on CapeOX for older patients with AGC have been conducted. A phase II study reported a median OS of 9.8–10.0 months, a median PFS of 5.6–6.0 months, and an ORR of 48.9–51.2% , 17. Hwawith AGC . In this control . These rThis study has several limitations. First, the single-arm phase II study design might limit the results to being exploratory. However, to our knowledge, this is the first prospective clinical trial for CapeOX among older patients with AGC in Japan. Second, following the ATTRACTION-4 and CheckMate 649 trials, nivolumab plus chemotherapy is now the standard first-line chemotherapy , 33. AltOlder cancer patients often exhibit decreased physical function, multiple comorbidities, and geriatric syndrome, so it is important to assess individual variability when weighing the risks and benefits of treatment. However, data on vulnerable older patients is limited, as the clinical trials for the development of the standard treatment have enrolled only a subset of older patients in good health who met the eligibility criteria. The method of evaluating the physical, mental, and social functions of patients, in addition to disease evaluation, is designated comprehensive geriatric assessment (CGA). The International Society of Geriatric Oncology recommends the use of CGA when assessing older patients with cancer to detect unaddressed problems and improve their functional status and possibly their survival . Notably2 b.i.d. on days 1–14 of each course) and oxaliplatin (100 mg/m2 on day 1 of each course), is effective and safe for Japanese older patients with AGC.Our phase II trial results suggest that CapeOX, comprising capecitabine (750 mg/m


In this study, we deployed a hydroponics-based experiment to explore the individual and mutual effects of different temperatures  and CO2 concentrations  on the uptake and translocation of sodium chloride  and cadmium nitrate (Cd(NO3)2·4H2O, 0.0, 0.2, 1.8, and 5.4 mg Cd/L) by rice seedlings. The results indicated that Cd and Na exposure significantly (P< 0.05) inhibited plant growth, but T2 and medium/high CO2 alleviated the effects of Cd and Na on plant growth. Neither significant synergistic nor antagonistic effects of Cd and Na were observed, particularly not at T1 or high CO2. At increasing temperatures, relative growth rates increased despite higher concentrations of Cd and Na in both rice roots and shoots. Similarly, higher CO2 stimulated the growth rate but resulted in significantly lower concentrations of Na, while the Cd concentration was highest at medium CO2. Coexposure experiments suggested that the concentration of Cd in roots slightly declined with additional Na and more at T2. Overall, our preliminary study suggested that global climate change may alter the distribution of mineral and toxic elements in rice plants as well as the tolerance of the plants.Plant kingdoms are facing increasingly harsh environmental challenges marked by the coexposure of salinity and pollution in the pedosphere and elevated CO The CO2 “fertilization effect” was observed by enhancing vegetation productivity  is one of the most important staple food crops worldwide, especially in eastern Asian countries. Over the last century, farmland deterioration due to metal contamination has become increasingly serious. Many anthropogenic activities, including mineral fertilizers, mining operations, manufacturing industries and landfills of industrial or domestic sludge, have resulted in an increasing amount of agricultural land being contaminated with heavy metals throughout the world  than each alone (Chenopodium quinoa Willd.) and reduced the shoot and root growth of experimental plants by more than 50%  to estimate the effects of temperature and CO2 concentrations on the plant growth of rice seedlings under salt (measured as Na) and Cd stress alone and in combination; (2) to investigate the individual and mutual effects of temperature and CO2 concentrations on the uptake and translocation of Na and Cd alone and in combination; and (3) to analyse the individual and mutual effects of temperature and CO2 concentrations on the metal distribution in rice tissues under Na and Cd stress alone and in combination.Because many biotic and abiotic factors are involved, it is difficult to quantify future trends. Present findings remain inconclusive, and very few studies have identified the effect of multiple stressors on terrestrial vegetation under the combined variation of temperature and CO22.1Oryza sativa L. cv. XZX 45, 2: 360 ppm). The modified 8692 nutrient solution was prepared in 1.0 L of water with KNO3 (285.2 mg/L), MgCl2·H2O (12 mg/L), CaCl2 (18 mg/L), MgSO4 (15 mg/L), KH2PO4 (33.46 mg/L), NaHCO3 (150 mg/L), H3BO3 (0.1855 mg/L), MnCl2 (0.415 mg/L), Fe-EDTA (4.83 mg/L), NaMoO4 (7 mg/L), CuSO4 (6.25 mg/L), ZnSO4 (2.99 mg/L), and CoCl2 (1.5 mg/L) (2 + 2 mM MES-Tris (pH 6.0)] for 4 h . The seeds (.5 mg/L) . After 1 for 4 h . FinallySolution A (Cd stress): Selected seedlings were exposed to a 50 mL Erlenmeyer flask spiked with 50 mL of Cd at nominal concentrations ;Solution B (Na stress): Selected seedlings were exposed to a 50 mL Erlenmeyer flask spiked with 50 mL of Na at nominal concentrations ;Solution C (Cd+Na stress): Selected seedlings were exposed to a 50 mL Erlenmeyer flask spiked with 50 mL of Cd+Na at nominal concentrations .3)2·4H2O, CAS: 10325-94-7] and sodium chloride , guaranteed reagents, were used in this study. A remarkable (P< 0.05) increase in IR was observed in rice seedlings from both Cd and Na treatments with different nominal initial concentrations . Four independent biological replicates were deployed at each Cd-/Na-/Cd+Na-treated concentration in the 3-d experiment. Each independent biological replicate contained ten rice seedlings of similar size. The selection criteria for exposure concentrations of Cd and Na are presented in Section 2.5 and Note that cadmium nitrate [Cd was obtained from Shaoguan Taihong Medical Appliance Co., Ltd. Three different levels of CO2 at 360 (ambient), 450 (medium) and 700 ppm (high) were designed. The testing temperature was set at either 23°C or 27°C. The relative humidity in the CCEC was 60 ± 2%. Liquid CO2 was stored in a tank connected to the computer-controlled commercial CO2 injection system with a PID controller that served as the CO2 source. The discharge of CO2 in the CCEC was manually regulated by a needle valve that was connected to mechanical flow meters. The CO2 concentration in the CCEC was a real-time detection and recorded by a computer. The resolution of CO2 levels in this CCEC is 1 ppm. Four treatments and their abbreviations were as follows:A COControl A (T1 + ambient CO2): rice seedlings were exposed to Solution A, Solution B and Solution C for 3 days and incubated in CCEC with a level of CO2 at 360 ppm (ambient CO2) and a temperature of 23°C;Treatment 1A (T1 + medium CO2): rice seedlings were exposed to Solution A, Solution B and Solution C for 3 days and incubated in CCEC with a level of CO2 at 450 ppm (medium CO2) and a temperature of 23°C;Treatment 2A (T1 + high CO2): rice seedlings were exposed to Solution A, Solution B and Solution C for 3 days and incubated in CCEC with a level of CO2 at 700 ppm (high CO2) and a temperature of 23°C.Control B (T2 + ambient CO2): rice seedlings were exposed to Solution A, Solution B and Solution C for 3 days and incubated in CCEC with a level of CO2 at 360 ppm (ambient CO2) and temperature at 27°C;Treatment 1B (T2 + medium CO2): rice seedlings were exposed to Solution A, Solution B and Solution C for 3 days and incubated in CCEC with a level of CO2 at 450 ppm (medium CO2) and a temperature of 27°C;Treatment 2B (T2 + high CO2): rice seedlings were exposed to Solution A, Solution B and Solution C for 3 days and incubated in CCEC with a level of CO2 at 700 ppm (high CO2) and a temperature of 27°C.2.3The relative growth rate (RGR) is one of the most important parameters reflecting the overall performance of plant physiology under environmental abuse . The metW(I) and W(F) are the initial and final fresh weights of rice seedlings, respectively.where 2.4The inhibition rate  of RGR was calculated using the following equation:C is the concentration of Cd and Na, t is the exposure period (d), RGR is the relative growth rate, i is replicate 1, 2,…, n and j is control 1, 2,…, m.where 2.510 transformed. Linear regression between log10Cd or Log10Na and IR (%) was plotted to calculate the concentrations of EC20, EC50 and EC75, which refer to the inhibition of 20%, 50%, and 75% of the RGR at 360 ppm CO2 and 25°C in comparison to the control, which were used as nominal initial concentrations for the tests with varying temperature and CO2 concentrations. More detailed information is presented in The effective concentration (EC) is the dose of pollutants that resulted in inhibitory effects on the possible parameters selected. In this study, the IR of RGR was used as a sensitive endpoint to estimate the EC value for Cd and Na treatments. The nominal concentrations of Cd treatments were 0, 0.25, 0.50, 1.00, 2.00, 4.00 and 8.00 mg Cd/L, while the nominal concentrations of Na treatments were 0, 0.1, 0.3, 0.5, 0.9, 1.2, and 1.6 g Na/L. For estimation of EC values, initial concentrations of Cd and Na were log2.63-HClO4 (v/v: 4:1) solution. The content of total Cd and Na in rice tissues from different treatments was determined by using inductively coupled plasma-atomic emission spectrometry .After 3 d of exposure, rice seedlings were collected and rinsed with distilled water and placed in a pretreated solution containing MES-Tris buffer (pH=6.0) for 4 h to remove all additional ions from the root surface and from the apparent free space. Then, rice seedlings were divided into roots and shoots. The remaining procedure was identical to our previous work . Dried p2.7P< 0.05). The significance of correlations was judged using tabled values for critical r (significance level α was 0.01 or 0.05).All data obtained were subjected to analysis of variance (ANOVA) and Tukey’s multiple range test at a significance level α of 0.01 or 0.05 between the treatments and control , respectively. Under Cd stress and T1 (T2), the RGRs of both ambient/medium/high CO2-treated rice seedlings ranged between 5.43-21.07%, 6.97-23.27%, and 8.06-24.58% , respectively. Under Na+Cd stress and T1 (T2), the RGRs of both ambient/medium/high CO2-treated rice seedlings ranged between 6.55-21.07%, 7.14-23.27%, and 8.09-24.58% , respectively. Apparently, for all treatments, the growth is positive, i.e., RGR is positive. In all cases, growth was significantly better at 27°C (P< 0.05) and at higher CO2. Growth inhibition of (approximately) 25%, 50% and 75% was observed for salt treatments with Na at 0.2, 0.6 and 1.1 g/L, respectively, and with Cd at 0.2, 1.8, and 5.4 mg/L, respectively. At 23°C, the effect of Na and Cd applied simultaneously was not significantly different from that of individual treatments. At 27°C, a slight depression of growth can be noticed in the combined treatment in relation to the single treatment with Na or Cd only.The measured RGRs for the Na treatments , Cd treatments , and Na + Cd treatments at T1 (23°C) and T2 (27°C) are shown in 3.22 treatments, the concentrations of Cd in rice shoots (roots) with Cd stress ranged between 4.97-13.21 μg/g DW (95.31-1787.23 μg/g DW), while the concentrations of Cd in rice shoots (roots) with Na+Cd stress ranged between 3.80-25.93 μg/g DW (107.5-1411.33 μg/g DW). Under T1 and medium CO2 treatments, the concentrations of Cd in rice shoots (roots) with Cd stress ranged between 7.31-27.66 μg/g DW (94.46-2652.8 μg/g DW), while the concentrations of Cd in rice shoots (roots) with Na+Cd stress ranged between 8.35-31.25 μg/g DW (126.5-2928.09 μg/g DW). Under the T1 and high CO2 treatments, the concentrations of Cd in rice shoots (roots) with Cd stress ranged between 7.02-22.67 μg/g DW (95.15-1852.19 μg/g DW), while the concentrations of Cd in rice shoots (roots) with Na+Cd stress ranged between 6.14-22.50 μg/g DW (126.77-1574.02 μg/g DW).The measured Cd content in rice shoots and roots separated for T1 (23°C) and T2 (27°C) is shown in 2 treatments, the concentrations of Cd in rice shoots (roots) with Cd stress ranged between 7.21-30.55 μg/g DW (97.81-2267.69 μg/g DW), while the concentrations of Cd in rice shoots (roots) with Na+Cd stress ranged between 5.55-29.33 μg/g DW (113.65-1474.37 μg/g DW). Under T2 and medium CO2 treatments, the concentrations of Cd in rice shoots (roots) with Cd stress ranged between 8.84-30.43 μg/g DW (127.72-3191.39 μg/g DW), while the concentrations of Cd in rice shoots (roots) with Na+Cd stress ranged between 9.09-29.61 μg/g DW (131.20-2387.40 μg/g DW). Under T2 and high CO2 treatments, the concentrations of Cd in rice shoots (roots) with Cd stress ranged between 9.38-28.23 μg/g DW (110.39-2955.65 μg/g DW), while the concentrations of Cd in rice shoots (roots) with Na+Cd stress ranged between 8.18-30.95 μg/g DW (126.13-1970.16 μg/g DW).Under T2 and ambient COP< 0.05). At 23°C, there is a trend toward high Cd concentrations in shoots when coexposed to Na and at low and medium CO2 (360 and 450 ppm), but this is not seen at 700 ppm CO2 and not at 27°C. At 23°C, the highest Cd concentration is found at 450 ppm CO2. Again, this trend is not apparent at 27°C, where concentrations of Cd are rather equal for all levels of CO2. As growth is best at 700 ppm CO2 and at 27°C, it can be concluded that the total uptake (mass of Cd) must be highest for these conditions.Notably, the concentration of Cd in shoots was much lower than that in roots. In shoots, higher concentrations were observed only at 27°C  with Na stress ranged between 3011.61-11655.06 μg/g DW (7569.71-12545.83 μg/g DW), while the concentrations of Na in rice shoots (roots) with Na+Cd stress ranged between 2813.5-11340.46 μg/g DW (7453.15-12805.63 μg/g DW). Under T1 and medium CO2 treatments, the concentrations of Na in rice shoots (roots) with Na stress ranged between 2568.85-8641.81 μg/g DW (5500.33-10866.99 μg/g DW), while the concentrations of Na in rice shoots (roots) with Na+Cd stress ranged between 2220.49-8589.50 μg/g DW (6425.30-10252.57 μg/g DW). Under T1 and high CO2 treatments, the concentrations of Na in rice shoots (roots) with Na stress ranged between 2257.15-6848.62 μg/g DW (5067.89-9131.93 μg/g DW), while the concentrations of Na in rice shoots (roots) with Na+Cd stress ranged between 1825.34-5020.30 μg/g DW (6657.62-9354.65 μg/g DW).The measured concentrations of Na in rice shoots and roots separated for T1 (23°C) and T2 (27°C) are shown in 2 treatments, the concentrations of Na in rice shoots (roots) with Na stress ranged between 4004.82-13243.01 μg/g DW (8057.37-14898.06 μg/g DW), while the concentrations of Na in rice shoots (roots) with Na+Cd stress ranged between 2006.02-10301.83 μg/g DW (3867.43-13083.43 μg/g DW). Under T1 and medium CO2 treatments, the concentrations of Na in rice shoots (roots) with Na stress ranged between 2607.55-9115.69 μg/g DW (6500.54-8351.60 μg/g DW), while the concentrations of Na in rice shoots (roots) with Na+Cd stress ranged between 2457.76-8309.92 μg/g DW (6459.82-12013.72 μg/g DW). Under T1 and high CO2 treatments, the concentrations of Na in rice shoots (roots) with Na stress ranged between 2635.73-8979.19 μg/g DW (5062.06-8811.56 μg/g DW), while the concentrations of Na in rice shoots (roots) with Na+Cd stress ranged between 2187.13-7068.07 μg/g DW (4223.16-7396.10 μg/g DW).Under T1 and ambient CO2 levels.Overall, Na concentrations in roots are higher than in shoots, but the difference is small. The maximum concentrations in roots reached 15 mg/g DW, and those in shoots were >12 mg/g DW. There is little difference in concentrations with/without additional Cd. There is also little difference between concentrations at T1 (23°C) and T2 (27°C). However, for almost all variants, Na concentrations have a strong tendency to be lower at high CO3.42 on the translocation factors (TFs) of Cd and Na in rice seedlings is shown in 2 concentrations, T2 (27°C) promotes the translocation of Cd and Na in rice seedlings.The effect of temperature and CO2 generally promoted Cd translocation in rice seedlings under 0.2 mg Cd/L stress, but no significant effect under 1.8 and 5.4 mg Cd/L stress was observed. While medium and high CO2 have little effect on Na translocation in rice seedlings under 0.2 and 0.6 g Na/L stress, high CO2 inhibits Na translocation under 1.1 g Na/L stress. At 27°C, high CO2 had an apparent effect on Cd translocation in rice seedlings under 0.2, 0.6, and 1.1 g Na/L stress. While medium CO2 inhibits Na translocation in rice seedlings under 0.2, 0.6, and 1.1 g Na/L stress, high CO2 promotes Na translocation.In addition, at 23°C, medium and high CO3.52 is not apparent.The measured concentrations of Cd in plants exposed to Cd+Na versus those measured in plants exposed solely to Cd are shown in 2, but this is the case for both types of exposure (with/without Cd). Overall, both concentrations of Cd and Na in roots and shoots are surprisingly similar, almost independent from the single or combined exposure.The measured concentrations of Na in plants exposed to Cd+Na versus those measured in plants exposed solely to Na are shown in 44.12 in air increases water use efficiency and stimulates the growth of plants  simultaneously; thus, the findings presented here are quite unique. Cd and Na  are two toxic chemicals affecting plant growth and development due to their toxicity  and medium/high CO2 levels significantly increased the RGR of stressed plants compared to the T1 (23°C) and ambient CO2 levels. Therefore, we believe that increasing temperature can stimulate the activities of enzymes in plants, such as RuBisCO . In this study, we noticed that the content of Cd in rice roots and shoots under Cd stress increased with increasing temperature in all COn stream , with sun stream . In contn stream . Root imn stream . In line4.2.22 concentrations in air were different. The concentration of Cd in rice tissues and, in particular, in roots increased at 450 ppm CO2 but decreased at 700 ppm CO2. The concentration of Na in rice tissues significantly decreased with increasing CO2 concentration and was lowest at 700 ppm CO2. This suggests that the uptake and translocation of Cd and Na are affected differently by the variation in CO2. Indeed, elevated CO2 resulting in higher Cd concentrations in plant tissues has been observed in different plant species +ambient/medium/high CO2 conditions. Interestingly, the interaction was not very pronounced but observable. These results implied that the temperature should be considered when CO2 is used as the regulator in the phytoremediation of Cd/Na/Cd+Na pollution. In addition, we noticed that the concentration of Cd in roots was depressed by coexposure to Na and more at T2, while with T1, more Cd was translocated into shoots. These changes indicate that the uptake processes of Cd and Na in rice are mostly independent, and only minor interactive effects occurred during exposure to mixtures of Cd and Na. A plausible explanation is that both molecules have very different radii and different charges (Na+ versus Cd2+), and it is unlikely that the same transporter enzymes are involved. However, Cd and Na are both cations and might compete for electrically charged adsorption sites. This may be the mechanism behind the lower Cd concentration in roots when the Na concentration is high. The cytosolic ionic strength was 0.3 M, in xylem sap at 0.01 , proteins, and metabolites.This work clarifies the simultaneous effects of changing temperatures and COThe original contributions presented in the study are included in the article/Conceptualization, Methodology, Supervision, Writing-reviewing and editing, and Funding acquisition, ST and X-ZY. Writing-original draft preparation, and Visualization, Y-XF. Investigation, Data analysis, Visualization, PT, C-ZL, and QZ. All authors contributed to the article and approved the submitted version.


To overcome numerous health disorders, heterocyclic structures of synthetic or natural origin are utilized, and notably, the emergence of various side effects of existing drugs used for treatment or the resistance of disease-causing microorganisms renders drugs ineffective. Therefore, the discovery of potential therapeutic agents that utilize different modes of action is of utmost significance to circumvent these constraints. Pyrrolidines, pyrrolidine-alkaloids, and pyrrolidine-based hybrid molecules are present in many natural products and pharmacologically important agents. Their key roles in pharmacotherapy make them a versatile scaffold for designing and developing novel biologically active compounds and drug candidates. This review aims to provide an overview of recent advancements  in the exploration of pyrrolidine derivatives, emphasizing their significance as fundamental components of the skeletal structure. In contrast to previous reviews that have predominantly focused on a singular biological activity associated with these molecules, this review consolidates findings from various investigations encompassing a wide range of important activities  exhibited by pyrrolidine derivatives. This study is also anticipated to serve as a valuable resource for drug research and development endeavors, offering significant insights and guidance.  Furthermore, the therapeutic molecules with a pyrrolidine ring in their structures such as daridorexant 12 (insomnia), pacritinib 13 (JAK-2 inhibitor), and futibatinib 14 (FGFR-4 inhibitor) were approved by the FDA in 2022  and quantitative structure–activity relationship (QSAR) in the synthetic pathway . So, in N-benzoylthiourea, thiohydantoin, thiazole, imidazole, and indole  and Butyrylcholinesterase (BuChE) inhibition properties were compared with those of the reference tacrine. The most active compounds were found to be phenyl- and methyl-substituted 15g (IC50: 0.029 µM), 15h (IC50: 0.041 µM), and 16g (IC50: 0.087 µM) with an indole ring in their structures , and for the hCAII enzyme, with a Ki value of 5.14 ± 0.61 nM, approximately 26 times greater than that of the reference AZA (132.53 ± 7.44 nM)  .20, a potential bioactive compound for the treatment of cystic fibrosis (CF). In this sense, more sophisticated analogs 21 containing this polycycle 20 can be classified as promising drugs for this purpose. Some studies regarding herbicide and pesticide activities, as well as surveys for plant growth control, are under investigation , 22b (210 ± 20 nM), 22c (120 ± 10 nM), 22d (250 ± 20 nM), and 22e (270 ± 20 nM) inhibited E. coli DNA gyrase at concentrations similar to the standard novobiocin (IC50 = 170 nM). Notably, the 4-chlorophenyl group is present in the structures of three (22b–d) of these five active molecules. Likewise, compounds 22a, 22b, 22d, and 22e showed activity close to that of novobiocin  against E. coli and S. aureus topoisomerase IV, while compound 22c , containing 4-chlorophenyl substituents on the pyrrolidine ring and 5-hydroxymethyl furan moiety on the 1,2,4 oxadiazole group, was more active than the reference. Compound 22c could be considered the lead compound for future research.in vitro antidiabetic effects with those of vildagliptin as a control. Compounds 23a–d showed 56.32%, 44.29%, 49.62%, and 66.32% inhibition, respectively. Compound 23d with the 4-trifluorophenyl substitution on the 1,2,4-oxadiazole group exhibited the best inhibition (IC50: 11.32 ± 1.59 μM) against the DPP-IV enzyme among these compounds.24 showed an antiproliferative effect against HT-29 and SH-SY5Y cells, acetylcholinesterase inhibition activity, and antioxidant activity.Haemonchus contortus. Compounds 25a (2-CH3) and 25b (3-OCH3) were discovered to be promising candidates against xL3 motility (IC50 = 0.78 µM for each), whereas compound 25c (4-I) was identified as an efficient inhibitor of L4 development (IC50 = 3.2 µM). Moreover, these compounds exhibited good selectivity against mammalian epithelial cells with limited inhibition of cell proliferation at 50 mM.in vivo anticancer metastatic potential. Compound 26, which contains pyridine, piperazine, and pyrimidine, together with the pyrrolidine ring, was identified as a CXCR4 antagonist with strong binding affinity to the CXCR4 receptor (IC50 = 79 nM) and a capability to inhibit CXCL12-induced cytosolic calcium flux (IC50 = 0.25 nM). In addition, to its favorable in vitro safety profile, compound 7 also demonstrated significantly enhanced metabolic stability in human and rat liver microsomes, as well as remarkable efficacy in a mouse cancer metastasis model. According to the experimental findings, compound 7 appeared to be promising in the development of CXCR4 antagonists.E. coli and P. aeruginosa) and Gram-positive (S. aureus and B. subtilis) bacterial strains and compared with the reference antibiotic chloramphenicol. Compound 27a, containing a butyl substituent, had the same effect (MIC: 16 μg/mL) against the Gram-positive bacteria S. aureus and B. subtilis using chloramphenicol as the reference, whereas compound 27b, containing a propyl substituent, had the same effect (MIC: 16 μg/mL) against S. aureus bacterial strains using chloramphenicol as the reference. Antifungal activity studies were conducted against the Candida albicans strain using ketoconazole as a reference. Compound 27a (MIC: 32 μg/mL), which is also the most active compound against the C. albicans strain, could be considered a promising molecule for further studies.15c–f, bearing an indole moiety at the R2 position and several aromatic substituents at the R3 position in their structure, and compound 17, obtained by ring arrangement exhibited twice the activity (MIC: 62.5 μg/mL) of the reference ampicillin against the A. baumannii strain  was determined to be the most effective antifungal agent, randomly selected compounds 28a, 28d , and 28e  exhibited little to no toxicity against the Vero cell line and Galleria mellonella. In the invertebrate model of disseminated candidiasis, compounds 28b and 28c (-F) showed the highest effectiveness against biofilm formation in vitro and also exhibited activity in vivo. Mitochondrial damage (XTT assay) and decreased adherence to the TR-146 cell line at 46.05 µM demonstrated necrotic cell death via fungal membrane interaction of compound 28b.Since tetrazole derivatives are already known to have powerful antifungal properties, 29 (57% inhibition) showed the best inhibitory property against ALR2 on an in vitro model of diabetic retinopathy. Furthermore, compound 29 decreased cell death processes in the photoreceptor-like 661w hyperglycemic cell line and restored physiological levels of oxidative stress, decreasing ALR2-associated ROS production.N-acylethanolamine acid amidase (NAAA), one of the key enzymes involved in the degradation of fatty acid ethanolamides (FAEs), specifically palmitoylethanolamide (PEA). Compounds 30a , 30b (4-(cyclohexyloxy)phenyl); IC50: 0.7 ± 0.2 μM), 30c , and 31  displayed potent inhibitory activity toward NAAA but had no such effect toward FAAH. Compound 31 showed good selectivity (IC50 for NAAA: 1.5 ± 0.22 μM and for FAAH: 550 ± 70 μM) with the strongest inhibition by reversible and competitive mechanisms against the NAAA enzyme as well as high in vitro anti-inflammatory activity in the lipopolysaccharide (LPS)-induced acute lung injury (ALI) model.32a (IC50 = 102 ± 31.4 and 162 ± 10.5 nM) and 32b (IC50 = 182 ± 25.2 and 242 ± 29.2 nM) with 4-phenoxyphenylsulfonyl groups in their structures inhibited MMP-2 and MMP-9 enzymes better than control LY52 (IC50 = 266 ± 19.2 and 360 ± 30.1 nM). Meta-substituted benzofuroxane molecules exhibited more inhibitory activity than the ortho-substituted molecules. Furthermore, compounds were tested against A549, ES-2, HeLa, K562, MCF-7, and MDA-MB-231 cancer cells, and the findings were compared to those of the positive control LY52. Compound 32a, which demonstrated the highest enzyme activity, also demonstrated an antiproliferative effect against HeLa cells, roughly 60 times greater than that of the control LY52 with an IC50 value of 3.82 ± 0.11 µM. All compounds, but 32a (34.43 μM/L) in particular, released moderate quantities of NO.N-benzoylthiourea-pyrrolidine derivatives containing N-phenyl 16b and N-methyl 16d groups were found to be four times more active against the A. baumannii strain and twice more active against A. hydrophila than the reference. Compound 16c, containing the 4-chlorophenyl group, showed better antituberculosis (anti-TB) activity than the reference ethambutol. Compounds 15a and 15b, which are among the pyrrolidine derivatives used in the synthesis of the corresponding compounds, also showed four times higher activity (MIC: 31.25 μg/mL) against A. baumannii than the reference ampicillin (MIC: 125 μg/mL)  .35a–d and the compounds 33a–d and 34a–d, used in the synthesis to access these molecules 35, were tested against Mycobacterium tuberculosis using the microplate Alamar Blue assay (MABA) method. Compared to the reference isoniazid and ethambutol, the synthesized compounds did not exhibit significant anti-TB activity, while the activities of the compounds were found in the range of 62.5–125 μg/mL against the Mycobacterium tuberculosis H37Rv strain (The anti-TB activities of the thiohydantoin-pyrrolidine derivatives v strain .33e (MIC: 1.95 μg/mL), which is one of the pyrrolidines synthesized for the preparation of N-benzoylthiourea derivatives used in the synthesis of thiazole-pyrrolidine derivatives and contains 4-chlorophenyl and indole groups in its structure, showed better anti-TB activity against Mycobacterium tuberculosis than the reference ethambutol (5 and 10 μg/mL). The anti-TB activity of compound 36 (MIC: 0.24 μg/mL), which was obtained by condensation of compound 33e with benzoyl isothiocyanate, was found to be eight times more potent than that of the starting compound 34e and better than that of the reference anti-TB drugs isoniazid and ethambutol. In addition, the anti-TB activity of compound 36, obtained by the addition of 4-bromomethoxy acetophenone to compound 34e, was found to be 0.48 μg/mL. Moreover, the antimicrobial activities of the compounds were tested against S. aureus, E. coli, A. baumannii, B. subtilis, and A. hydrophila strains. Compound 15c, which contains an indole heterocycle, together with a fused bicyclic pyrrolidine ring, was twice more active against A. baumannii than the reference antibacterial agent ampicillin (MIC: 125 μg/mL) with a MIC value of 62.5 μg/mL , CH1 (human ovarian carcinoma), and SW480 (colon cancer) cancer cell lines and noncancerous murine embryonal fibroblasts (NIH/3T3), and studied the cell death mechanism using the flow cytometry method. The outcomes were evaluated against a cisplatin control group. The thiosemicarbazone pyrrolidine–copper(II) complexes, with two pyrrolidine rings in their structure, were the most potent anticancer compounds against three cancer lines among the synthesized ligands and complexes. Copper complex 37a (IC50: 0.99 ± 0.09) was approximately three times more potent than cisplatin used as a control (IC50: 3.5 ± 0.3) against the SW480 cancer cell line, while copper complex 37b (IC50: 3.7 ± 0.1) showed approximate activity to cisplatin. The results of the research revealed that altering the coordinating groups of the ligands enhanced their antiproliferative activity.in silico EGFR potential as well as their antibacterial and antifungal activities. Antibacterial activity studies were compared with those of reference cefaclor against S. aureus, E. coli, and P. aeruginosa bacteria. Compound 38, which contains two nitrophenyls together with the pyran ring as a substituent in its structure, showed the best activity against three bacteria . Antifungal activity studies were also performed against A. niger and C. albicans fungal strains using ketoconazole as the reference. Compound 38 also showed the highest activity against the respective fungal strains [A. niger (MIC: 1.78 μg/mL) and C. albicans (MIC: 3.18 μg/mL)]. Compared to the reference, in silico studies showed that the compounds could serve as EGFR inhibitors.R,2S)-2-fluorocyclopropyl]naphthyridone derivatives containing oxime-functionalized pyrrolidine and evaluated their antimicrobial activities. The antituberculosis (TB) activity of pyrrolidine derivatives was compared with that of the reference drugs isoniazid and rifampicin using the MABA method against the Mycobacterium tuberculosis (MTB) H37Rv ATCC 27294 (MTB-1) strain. Compounds 39a, 39c, and 39d, which contain 4-F, 4-CH3, and 2′,3′-methylenoxyl substituents, respectively, were found to be the most active compounds against the MTB-1 strain with a MIC value of <0.25 μg/mL. In addition, these most active compounds against the clinically isolated MDR-MTB 6133 (MTB-2) strain resistant to isoniazid and rifampicin showed much better anti-TB activity than the references . The antibacterial activity of the compounds was tested against a series of Gram-negative  and Gram-positive  strains. Ciprofloxacin (CPFX), levofloxacin (LVFX), and moxifloxacin (MXFX) were used as references. Compounds 39b, 39c, and 40a–c showed better activity (MIC: <0.008–32 μg/mL) against Gram-positive bacteria than against Gram-negative bacteria than the references (MIC: 0.125–>128 μg/mL).41 (Ki: 0.24 ± 0.06 µM) exhibited the highest activity in suppressing MDM2-mediated p53 degradation as well as GPX4 levels in MCF-7 breast cancer cells.42c (IC50: 1.59 ± 0.08 μg/mL), 42d (IC50: 1.67 ± 0.09 μg/mL), 42e (IC50: 1.63 ± 0.09 μg/mL), and 42f (IC50: 1.57 ± 0.10 μg/mL), bearing R1: NO2, H, H, and Br; R2: H, benzyl, benzyl, and H; and Ar: Br, F, Cl, and Cl substituents, respectively, showed approximately the same level of inhibition as the reference acarbose (IC50: 1.56 ± 0.09 μg/mL). Compound 42a (IC50: 1.49 ± 0.10 μg/mL), with a 4-bromo substituent on the isatin ring in its structure, was more active than the standard acarbose. In subsequent investigations of in vivo hypoglycemic activity, 42a–c and 42f were the most active compounds that decreased the blood glucose levels. The findings suggested that N-substituted groups attached to isatin and substituents on the phenyl ring may both affect enzyme function.43a (IC50: 0.85 ± 0.20 μg/mL) and 4-bromophenyl-substituted 43b (IC50: 0.80 ± 0.10 μg/mL) were discovered to be roughly 11 times more active against the HepG2 cell line than the reference (IC50: 9.00 ± 0.76 μg/mL) among the non-substituted oxindole compounds. In addition, compound 43a (IC50: 2.00 ± 0.60 μg/mL) was more active than the reference compound against the HCT-116 cell line, whereas compound 43b (IC50: 3.00 ± 0.50 μg/mL) displayed the same activity. One of the dibromo-substituted oxindole derivative compounds, 3-NO2-substituted 43c , showed higher activity against all three cell lines than the reference, as well as approximately twice better activity than cisplatin (IC50: 9.00 ± 0.29 μg/mL) against the MCF-7 cell line.44, which was found to be the most active antifungal agent after broad-spectrum antifungal screening. In the tests, compound 44 containing OCF3 and 2-Cl was found to be the most active compound against the C. albicans strain with a MIC value of 4 μg/mL. Compound 44 was then tested against pathogenic strains of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and Cryptococcus neoformans isolated from humans. These fluconazole-resistant organisms were inhibited by compound 44. In addition, compound 44 reduced the activity of the C. albicans strain, which gained resistance to antifungal agents by forming filaments and biofilms, by up to 25% at concentrations of 32 μg/mL and 64 μg/mL. The MTS assay was also used to determine the non-toxicity of compound 44 to the HCT116 mammalian cell line in vitro.E. coli and S. aureus and inhibitory potency against the AChE enzyme. The most active compounds against E. coli bacteria were compounds 45b (R1: Cl and R2: CH2Ph) and 27c . The compounds did not show significant activity against the bacterial strains. Compounds 45a  and 45d  showed the strongest inhibition against the AChE enzyme compared to the reference tacrine. The IC50 values of compounds 45a and 45d were found to be 69.07 ± 10.99 µM and 111.38 ± 10.11 µM, respectively.46a with a 4-Br substitution was the most active, whereas compound 46b with a 4-OCH3 substituent was the least active. Moreover, cell death was noted to be an apoptotic pathway mediated by the increased activation of caspase-3 proteins, while halogen-substituted pyrrolidines were found to be more active than the others.47a–g and 48a, b, known for their anticancer properties and containing a spirooxindole structure, by metal-catalyzed imine azomethine ylide cycloaddition reactions following by the Heck reaction.49c (IC50: 1.07 μg/mL), which contains a 2,4-dichlorophenyl group, was found to be approximately 12 times more active than the reference cycloserine (IC50: 12.47 μg/mL) and 36 times more active than the reference pyrimethamine (IC50: 37.35 μg/mL) against Mycobacterium tuberculosis H37Rv. In addition, compound 49a (IC50: 55.79 ± 2.60 μg/mL), containing a 4-fluorophenyl substituent, exhibited the same activity as the standard doxorubicin (IC50: 55.91 ± 1.34 μg/mL) against CCRF-CM cell lines but did not show significant activity against HT29 and MCF7 cell lines. Moreover, compound 49b (IC50: 1.16 ± 0.1 μmol/L), containing 4-methoxyphenyl, was the most active compound against the AChE enzyme compared to the reference donepezil.Culex quinquefasciatus and compared with the reference permethrin. Compounds 50a (LD50 = 26.06 μg/mL), containing acetamide in its structure, and 50b (LD50: 26.89 μg/mL), containing a benzamide moiety, showed larvicidal activity comparable to the reference (LD50 = 26.14 μg/mL). The experimental results and the highest docking scores for compounds 50a and 50b against the 3OGN protein are consistent.. The antibacterial activities of the compounds were tested using the microdilution method against E. coli, S. typhimurium, B. cereus, and S. aureus strains, and their cytotoxicity was tested on L929 cells using the XTT method. Compound 51a (MIC: 21.70 ± 0.36 and 30.53 ± 0.42 μg/mL), containing the 4-fluorophenyl substituent attached to the thiazole ring, was compared with the reference gentamicin (MIC: 22.65 ± 0.21 and 22.17 ± 0.47 μg/mL), showing the best activity against B. cereus and S. aureus. In addition, compounds 51a, b, containing 4-bromophenyl and 4-fluorophenyl substituents, respectively, did not show toxicity at the concentrations performed.Echinochloa crus-galli) and rape (Brassica campestris) model plants. Compounds 52a (84%), with a 4-CH3 substituent, and 52b (65.6%), with 2-OCH3, showed the highest inhibition rates.in vivo in animal models using MES, scPTZ, and psychomotor tests (6 Hz). The effects of compounds 53, which include 2-morpholinoethyl, 3-morpholinopropyl, 3-CF3Ph, and 3,4-dichlorophenyl substituents in their structures, on GABA transporters, sodium, and calcium channels were studied to determine the anticonvulsant action mechanisms. Compound 53b was the most active compound, with an ED50 of 62.14 mg/kg in the MES test and 75.59 mg/kg in the 6 Hz test compared to the reference valproic acid and ethosuximide. In addition, the hot plate and writhing tests evaluated the antinociceptive activities of these active compounds. The results showed that compound 53d demonstrated an analgesic effect comparable to that of aspirin at a dose of 30 mg/kg in the writhing test, whereas compounds 53c and 53d demonstrated central analgesic activity at the same dose in the hot plate test.54a (IC50: 0.05 µΜ), 54b (IC50: 0.12 µΜ), and 54c (IC50: 0.18 µΜ) , bearing benzyl, 4-fluorobenzyl, and benzyl, respectively, and 54d (IC50: 0.035 µΜ), which are boronic acid derivatives, could be considered the most potent ATX inhibitors. Compound 54d (IC50: 0.035 µΜ) with S configuration was the most active among these compounds.55–58 . It is an antibiotic isolated from various Streptomyces species . This naturally occurring monohydroxypyrrolidine alkaloid acts to inhibit bacterial protein synthesis. It is used as an antiparasitic, antimicrobial, antineoplastic, and antifungal agent and as a DNA and protein synthesis inhibitor (blocks peptide bond formation). Anisomycin 6 has also been used for the treatment of trichomoniasis and amoebic dysentery and has shown in vitro potential antitumor activity against human tumor cell lines , result from metabolic diversity and occur in phylogenetically distinct plant families that possess the tropane core structure  . The antaine 64] . Tropinoaine 64] .R)-bgugaine 65, (R)-irnidine 66, and (R)-irnidine 67] are versatile pyrrolidine alkaloids isolated from the tubers of Arisarum vulgare, which revealed significant bioactivities such as apoptosis, cytolysis effects, and antimycotic, antimitotic, antibacterial, and antifungal activities (R)-enantiomers of these alkylpyrrolidine alkaloids have been reported to possess a binding affinity for DNA, together with induced significant DNA damage in a human hepatoblastoma (HepG2) cell line - and -isomers of 2-carboxy-4-(5-carboxy-l-methyl-2-hexenylidene)-3-pyrrolidineacetic acid 74 after their structural determination  receptor agonist. Acromelic acid (C) was found to exhibit a lethal toxic effect on mice at a dose of 10 mg/kg  10 mg/kg .78 and emiline 79, were isolated from Jacobaea gigantea (Jacobaea/Senecio genera) (Asteraceae) by J. gigantea is also known as an endemic herb that was found to grow in Algeria, Morocco, and Tunisia.Recently, two tetracyclic functionalized pyrrolidine-type alkaloids, senecipyrrolidine 80, acanthophoraine B 81, and acanthophoraine C 82, were isolated from a red alga called Acanthophora spicifera, and their antibacterial activities were screened against the bacterial strains, including E. faecalis, S. aureus, P. aeruginosa, K. pneumoniae, A. baumannii, and E. coli, and no desirable effects were found on the tested bacteria , Schizanthus hookeri, Carallia brachiate, and genera Schizanthus and Erythroxyloncoca. These structurally simple 2-substituted naturally occurring pyrrolidine alkaloids are important pharmacophores due to their intriguing biological and pharmacological activity. Some of its derivatives have been found to show significant hallucinogenic characteristics and displayed antitrypanosomatid and antiplasmodial activities . The studies have shown that these two alkaloids exhibited weak cytotoxic activity against the murine lymphoma cancer cell (L5178Y) line.89–96 (Broussonetia species), mainly isolated from the branches of Broussonetia kazinoki Sieb. (Moraceae). Shibano et al. reported the isolation, structural determination, and bioactivities of these series of pyrrolidine alkaloids called broussonetines A–H 89–96 and screened them for their inhibitory activities on some glycosidases (There are many structurally identified polyhydroxy pyrrolidine alkaloids that possess glycosidase inhibitory properties, which have been isolated from natural sources . Some ofosidases .via molecular hybridization will contribute to the development of alternative molecules to drugs that are used in the treatment of diseases and have various limitations. The pyrrolidine skeleton continues to provide new formulas of synthetic and natural compounds with diverse biological activities. Many SAR studies have confirmed the crucial role of this heterocycle in the effective interaction with the natural target. It is noteworthy that, despite the overwhelming variety of substituents anchored to it, a selected number of them are ready to promote beneficial biological interaction. It is important not to discard minor effects in biomedical applications because the fine tuning of the atomic arrangement of the radicals bonded to the ring can raise a very important interaction. The chiral natural pool also provides new compounds, which can serve as starting points to design potent inhibitor agents and can also be useful for the inspiration for the search of many other drug surrogates. This field has wide scope, and extensive work is necessary to develop new drugs.This review focuses on the current progress in pyrrolidine derivatives, which show multiple pharmacological activities and functions in the treatment of several health disorders. Pyrrolidine-based therapeutic candidates are currently used in the therapy of some diseases. In view of the aforementioned information, combining pyrrolidine compounds, which are known to exhibit many biological activities, with different bioactive heterocyclic structures


Staphylococcus aureus phage ISP and Pseudomonas aeruginosa phages PNM, 14-1 and PT07 were evaluated. These phages were magistrally prepared by diluting active pharmaceutical products and storing them in different polypropylene/polycarbonate syringes at 4°C. Titers were monitored for 7–21 days using the double-agar overlay method. For 7 days, titers remained stable. Afterwards, the phage titer reduced for all phages with an average reduction of 0.78 log (P < 0.0001) plaque-forming units (PFUs)/mL after 14 days and 1 log PFU/mL after 21 days (P < 0.0001). PT07 titers dropped below the predefined threshold after 2 days in polycarbonate syringes. In addition, the stability after irrigation using a nasal douche (CRS) and infusion through a catheter (sepsis) was assessed. No changes were observed after using these devices with a mean change in titer of −0.07 log PFU/mL (P = 0.9062) and −0.03 log PFU/mL (P = 0.7350), respectively. In conclusion, a decreased titer after storage of a phage solution can lead to administration of subtherapeutic concentrations. In our experiments, we demonstrated that liquid phage solutions can be stored in syringes at 4°C for 5–7 days. PT07 appeared to be less stable, especially in polycarbonate syringes. Irrigation using a nasal douche or infusion through a catheter does not impact phage titer.Bacteriophages have (re)gained popularity as an adjunct antibacterial therapy. On top of the self-amplifying character of phages, their titer can be influenced by several factors which could lead to reduced titers after preparation and storage. The stability of phage preparations, used for the treatment of chronic rhinosinusitis (CRS), sepsis, pulmonary, and musculoskeletal infections, was investigated. As antimicrobial resistance becomes more prevalent, the application of (bacterio)phage therapy as an alternative treatment for difficult-to-treat infections is (re)gaining popularity. Over the past decade, numerous promising case reports and series have been published demonstrating the therapeutic potential of phage therapy. However, important questions remain regarding the optimal treatment protocol and, unlike for medicinal products, there are currently no predefined quality standards for the stability of phage preparations. Phage titers can be influenced by several factors which could lead to reduced titers after preparation and storage and, ultimately, subtherapeutic applications. Determining the stability of different phages in different recipients according to the route of administration is therefore one of the first important steps in establishing a standardized protocol for phage therapy. S. aureus directed phage which covers ~73% of clinical isolates phages as magistral preparations was approved in Belgium . CurrentThe stability of phage solutions can be influenced by several factors including phage morphology, formulation  , 5, temp9 (SD ± 9.5×108), 6.1 × 109 (SD ± 2.4 × 109), 8.4 × 109 (SD ± 1.9 × 108) and 2.7 × 109 (SD ± 1.4 × 109) plaque-forming units (PFU)/mL was prepared for ISP, 14-1, PT07, and PNM, respectively. After 7 days, no significant reduction in ISP , 14-1 , and PNM  titers was observed (9 PFU/mL after 2 days with a mean reduction of 0.760 log PFU/mL after 7 days (P < 0.0001).To determine the stability of 1 mL phage solutions for intranasal applications in patients with difficult-to-treat CRS, the phage titer was measured repeatedly for 7 days . A solutobserved ; Table 17 (SD ± 3.4 × 107) for ISP, 7.0 × 107 (SD ± 1.5 × 107) for 14-1, 5.1 × 107 (SD ± 1.1 × 107) for PT07, and 2.6 × 107 (SD ± 1.1 × 107) PFU/mL for PNM. Over 7 days, the titers remained stable for PT07  and 14-1 , whereas a significant reduction was observed after 7 days for ISP  and PNM . All phage titers remained above the ETT of 1 × 107 log PFU/mL , 2.4 × 108 (SD ± 2.4 × 108), 1.3 × 108 (SD ± 3.9 × 107), and 1.8 × 108 (SD ± 6.9 × 107) PFU/mL were prepared for ISP, 14-1, PT07, and PNM, respectively. The first 7 days, the titer remained stable, except for PT07 . After 7 days, the titer of all phages dropped below the ETT of 1 × 108 PFU/mL and significantly lower titers were observed compared to baseline for ISP , PT07 (mean change = −0.618 log PFU/mL), and PNM . After 21 days, a mean reduction of −0.646 (P = 0.6301), –0.748 (P = 0.4267), –0.863 (P = 0.1732), and −1.5 log PFU/mL (P = 0.0847) was observed for ISP, 14-1, PT07, and PNM, respectively  for ISP, 1.8 × 108 (SD ± 4.6 × 107) for 14-1, 2.0 × 108 (SD ± 9.7 × 107) for PT07, and 4.7 × 108 PFU/mL (SD ± 1.6 × 107) for PNM were prepared. After 7 days, a significant reduction in titer of 14-1  and PNM  was observed, but all titers remained above the ETT of 1 × 108 PFU/mL during the entire experiment  and after storage at room temperature for 1 h   .P = 0.9350), 14-1 , PT07 , and PNM  remained stable and above the ETT  infusion of 50 mL through a three-lumen catheter for 5 min. After infusion, the titers of ISP  of ISP, 14-1, PNM, and PT07, respectively, was retrieved from the polypropylene, indicating that a high number of phages attach to the plastic . Duyvejock et al. demonstrated that storage of PNM, ISP, and 14-1 phage at 107 PFU/mL resulted in a major decrease in phage titer after 14 days, independent of phage morphology or storage solutions  through a catheter.Based on these findings, it is clear that stability testing is of key importance before application of phage preparations in patients, and this should be optimized for each specific phage, storage solution, recipient, and application method. Ideally, these measurements are predefined by regulatory agencies, as is the case for classic medicinal products. This could be arranged on a national level, but—to promote standardization—preferably on a European level, whereby scientific organizations such as the recently formed ESCMID Study Group for Non-traditional Antibacterial Therapy (ESGNTA) could play an important role . We suggThe main limitation of this study is that only phages that are available as APIs in Belgium, with predefined titers and stored in specific storage recipients were tested. No comparison was made between several phage concentrations or storage recipients and no evaluation of phage-phage interactions was performed. We also evaluated the phage stability of solutions with a fixed pH of 5.5 that is used in our clinical setting, and we did not test a range of pH values. Future studies should investigate these factors contributing to (in)stability of phage preparations. Additionally, the adhesion of phage particles to polymer materials (including syringes and catheters) could be further evaluated, for instance using scanning electron microscopy, as indicated by Richter et al. .The treatment protocol, including application method, dosage, titer, and volume, for each indication was carefully designed after multidisciplinary consensus by the CBL, in the context of the PHAGEFORCE project .At our center, phage therapy is applied locally for each treatment indication, except for sepsis . For CRS7 PFU/mL active phages before application in patients with CRS and MSI. For CF/Bx and sepsis, a titer of 108 PFU/mL was chosen, to anticipate for a reduced titer after inhalation through a nebulizer (CF/Bx), and intravenous distribution (sepsis). These titers were defined as ETT. As no literature is available on the optimal dosage, the titer was based on years of experience by the Eliava Institute and the general rule that phage preparations should contain at least 106–107 PFU/mL phage particles (The CBL came to a consensus that a phage solution should contain 10articles .S. aureus (ISP) and three P. aeruginosa  phages, was made based on the predominance of S. aureus and P. aeruginosa infections in the indications that are treated in our center , prior to administration to patients.10, 5.1 × 109, 2.0 × 109, and 1.0 × 1010 PFU/mL, respectively, and pH ranging between 6.0 and 8.0 according to the conformity reports of Sciensano. The API solutions were transported in glass vials  with a titer of 1.3 × 10ss vials .9 PFU/mL were prepared in 1 mL polycarbonate syringes . In a later stage, as previously described, the phage solution is diluted with 99 mL NaCl 0.75%/sodium bicarbonate 0.25% mixture by the patient to obtain a final titer of 107 PFU/mL. For MSI, 20 mL solutions at 107 PFU/mL were prepared in 20 mL polypropylene syringes . In view of CF/Bx applications, using a vibrating mesh nebulizer, 4 mL solutions with a titer of 108 PFU/mL were prepared in 5 mL polypropylene syringes . Finally, a 50 mL solution with a titer of 108 PFU/mL was prepared and stored in a 50 mL polypropylene syringe  for intravenous use in septic patients. Syringes were stored at 4°C . Next, 100 µL of the diluted phage solution was mixed with 200 µL of overnight grown host bacterium  and 4 mL of lysogeny broth soft agar (0.7%). The mixture was dispersed evenly on solid tryptic soy agar plates (bioTRADING Benelux B.V.). After overnight incubation at 37°C, translucent plaques were counted and the titer (PFU/mL) was calculated, as previously described. Titers of each sample were determined in triplicate.First, tenfold serial dilutions were prepared in phage buffer  was diluted with 99 mL NaCl 0.75%/sodium bicarbonate 0.25% mixture (37°C) in a plastic nasal douche . The titer was assessed at baseline (T−1), after squeezing the bottle immediately after dilution (T0) and after storing for 60 min at room temperature (T1). For sepsis, the stability after infusion through a three-lumen central venous catheter  was determined by measuring the titer at baseline (T0) and after infusion for 5 min (T1).For CRS, a mechanical test was performed to determine the impact of squeezing the nasal bottle manually, and thus applying positive pressure. A 1-mL phage solution  was tested on day 7. Syringes containing the phage solution were completely emptied and the number of active phages (PFU and PFU/mL) at baseline was determined. Afterwards, Tween 20 (0.1%) was added to the syringe, after which it was placed on a shaking platform at 4°C for 45 min. The number of active phages  was calculated. This process was repeated for three cycles. Tween 20 (polysorbate 20) is a nonionic detergent that is widely used as a solubilizing agent, stabilizer, and washing agent. It acts by modifying the integrity and structure of surface and free-floating vesicles, increasing the hydrophilicity of the surface, improving surface passivation, and inhibiting the nonspecific binding between biomolecules and the surface . The dett test and Wilcoxon test, respectively. Additionally, differences between more than two variables were measured using the one-way analysis of variance (ANOVA), for paired data with normal distribution, or the Friedman test, for paired data without normal distribution. Multiple testing was corrected with the built-in Dunnett test for one-way ANOVA. Normality was determined using the Shapiro-Wilk test. P values <0.05 were considered statistically significant and 95% confidence intervals (CIs) were calculated.Statistical analysis was performed using GraphPad Prism (version 9.4.1.). For each time point, the titer was assessed in triplicate and mean and standard deviation were calculated. Differences in continuous data, with/without normal distribution, were assessed using a paired Stability testing is of key importance before human application of magistral phage preparations. A decreased titer after storage of a phage solution can lead to the administration of subtherapeutic concentrations. In our experiments, we demonstrated that phage solutions can be safely stored in polypropylene/polycarbonate syringes at 4°C for a maximum of 7 days. Afterward, the stability of the titer above the predefined ETT cannot be guaranteed. Phage PT07 seemed to be less stable over time, especially in polycarbonate syringes. In addition, phage titers remained stable after rinsing with a nasal douche and irrigation through a catheter.


Orthosteric inhibition of kinases has been challenging due to the conserved active site architecture of kinases and emergence of resistance mutants. Simultaneous inhibition of distant orthosteric and allosteric sites, which we refer to as “double-drugging”, has recently been shown to be effective in overcoming drug resistance. However, detailed biophysical characterization of the cooperative nature between orthosteric and allosteric modulators has not been undertaken. Here, we provide a quantitative framework for double-drugging of kinases employing isothermal titration calorimetry, Förster resonance energy transfer, coupled-enzyme assays, and X-ray crystallography. We discern positive and negative cooperativity for Aurora A kinase (AurA) and Abelson kinase (Abl) with different combinations of orthosteric and allosteric modulators. We find that a conformational equilibrium shift is the main principle governing this cooperative effect. Notably, for both kinases, we find a synergistic decrease of the required orthosteric and allosteric drug dosages when used in combination to inhibit kinase activities to clinically relevant inhibition levels. X-ray crystal structures of the double-drugged kinase complexes reveal the molecular principles underlying the cooperative nature of double-drugging AurA and Abl with orthosteric and allosteric inhibitors. Finally, we observe the first fully-closed conformation of Abl when bound to a pair of positively cooperative orthosteric and allosteric modulators, shedding light onto the puzzling abnormality of previously solved closed Abl structures. Collectively, our data provide mechanistic and structural insights into rational design and evaluation of double-drugging strategies. Protein allostery is one of the fundamental regulatory mechanisms involved in various biological processes . Specifiin vitro and in vivo , asciminib, has been highly effective in inhibiting Abl  in vivo –12. Rema in vivo . Conside in vivo , 16. HowHerein, we provide the quantitative framework for double-drugging using two targets: Aurora A kinase (AurA) and Abelson kinase (Abl). Both kinases participate in various cellular pathways, and their dysregulation results in a multitude of cancers, such as breast cancer and leukemia –19. Comm50 = 13 nM, Ki = 0.87 ± 1.44 nM . Since  1.44 nM , 28, we  1.44 nM . Alignin 1.44 nM . To test 1.44 nM . IdenticKeq= 0.67  Mb2 is an inhibiting monobody that we had obtained an X-ray crystal structure in complex with AurA, ii) Mb3 exhibits larger inhibition than Mb2, and iii) AurA-Mb3 complex exists in a monomeric form unlike the dimeric AurA-Mb2 complex . We indeeq= 0.67 ) and asseq= 0.67 . Thus, aeq= 0.67 . We speceq= 0.67 , 28.To confirm whether the mechanism of cooperativity between Mbs and danusertib follows a classic allosteric model, we tested binding of Mb6 to the AurA-danusertib complex. Despite high affinity, Mb6 binding does not change AurA’s activity, implying that Mb6 binding does not shift the active/inactive conformational equilibrium of AurA . Indeed,For a reversible two-state allosteric model, the same fold-change of cooperativity must be observed when reversing the order of binding. To measure changes in the affinity of danusertib upon Mb binding, we had to employ competitive replacement ITC with adenosine 5’- diphosphate (AMPCP), since danusertib binds too tightly to AurA for direct measurement. Indeed, the measured cooperativities are matching quantitatively regardless of the binding order .Next, we probed biological relevance of these observed cooperativities by measuring the inhibition of AurA kinase activity using Lats2 peptide as a substrate with cellular ATP concentrations. Pre-incubation of inhibiting Mbs resulted in a vastly decreased amount of danusertib required to cause 90% inhibition of AurA activity . This coWe solved X-ray crystal structures of double-drugged AurA complexes to further understand the structural features responsible for the positive and negative cooperativity between Mbs and danusertib . The comO-27 of danusertib’s methoxy moiety than with E181 in the MD simulation, which is the state sampled in our crystal structure as well , we scrutinized the thermodynamic parameters of our ITC studies on danusertib binding to different AurA-Mb complexes –D. We fi as well .In the inactive conformations of AurA, the broken K162-E181 salt-bridge stems from the α-C helix and DFG-motif being positioned in the “out” conformation, such that F275 positions between K162 and E181 (PDB: 4C3R and 2J50)  Fig. 2G. Thus, wIntrigued by our mechanistic insights into double-drugging of AurA, we turned to Abl, the only target currently in clinical trials for double-drugging. It has been shown that the combination of the orthosteric inhibitor imatinib and the allosteric inhibitor asciminib abolishes the emergence of resistance mutations , 32–34, Abl exists in a conformational equilibrium between open, active and closed, inactive conformations , 35, 36 In ITC experiments, we find that imatinib binds 5-fold tighter to AblKD, which exists exclusively in the open conformation, than to AblFL . This coImportantly, we measure identical negative cooperativities between imatinib and asciminib on both AblKD and AblFL, regardless of binding order, within the range of errors . Due to 50 = 44 nM) . We hypothesized SKI would have the same effect on Abl due to its structural homology with Src kinase. Since SKI binding to Abl did not result in a detectable heat change in ITC, we turned to FRET experiments to quantify this interaction , an orthosteric inhibitor that tightly binds to Src kinase (IC= 44 nM) , 42, beceraction , Fig. S9eraction .Interestingly, it had been reported that allosteric inhibitors of Abl other than asciminib  actually do not inhibit the catalytic activity despite binding to AblKD , 43, 44.50 for AblKD than for AblFL, while SKI exhibited a higher IC50 for AblKD than for AblFL. Second, pre-incubation of AblKD with asciminib increased the IC50 for imatinib. This negative cooperativity arises from binding preferences of imatinib and asciminib to opposite conformations. In contrast, double-drugging of AblKD with SKI and asciminib resulted in a reduced IC50 since both have a binding preference to the same, “closing competent” conformation, highlighting their positive cooperativity , and not via binding to its active site  compared to closed AblFL (Kd = 72.4 nM) and negative cooperativity between imatinib and asciminib buttress Grzesiek’s model where imatinib’s preferential binding to the open conformation of Abl arises from its orthosteric site binding with nanomolar affinity.To combat on-target cancer drug resistance, double-drugging holds promise to be a powerful strategy. The rationale behind is multiplication of individual resistance mutational probabilities for each drug. Impressively, combinations of asciminib and various orthosteric inhibitors, including imatinib, indeed abolish the emergence of Abl resistance mutants and this double-drugging of Abl is currently in clinical trials . Given thibitors . This cohibitors , 32. Ourive site . This is  in pET28a and LPP (#79748) from Addgene were co-transformed in BL21(DE3) cell and plated on Kan/Spec LB plate. Few robust colonies were grown overnight in 250 ml TB culture to inoculate a 1 L culture to OD = 0.2. 1 L TB culture was grown to OD = 0.6 – 0.8 and induced with 0.6 mM IPTG for 16 hours at 21 °C. Cells were centrifuged at 4500 rpm for 15 min and resuspended in Buffer A. Cells were sonicated in the presence of EDTA-free protease inhibitor cocktail, lysozyme and DNAse, and then spun down at 18000 rpm. Supernatant was filtered through 0.22 μm filtering unit and passed through Ni-NTA columns. The protein was eluted at 100% Buffer B and protein fractions were pooled and combined with TEV and GST-LPP, then dialyzed overnight against Buffer C at 4 °C. Cleaved Aurora A was passed through tandem Ni-NTA and GST columns to remove uncleaved Aurora A, cleaved His-tag, and GST-LPP, and ran through 26/600 S200 pg gel filtration column with Buffer D. Pure fractions were pooled and concentrated to around 40 μM, and stored in −80 °C.2, 10% glycerol, pH 8.0Buffer A: 50 mM Tris-HCl, 300 mM NaCl, 20 mM MgCl2, 10% glycerol, pH 8.0Buffer B: 50 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, 20 mM MgCl2, 5 mM TCEP, 10% glycerol, pH 7.5Buffer C: 50 mM Tris-HCl, 300 mM NaCl, 1 mM MnCl2, 5 mM TCEP, 10% glycerol, pH 7.5Buffer D: 20 mM Tris-HCl, 200 mM NaCl, 20 mM MgClet al.  were purified with on-column refolding as described in Zorba et al. .Plasmids (kanamycin-resistance) encoding AblKD  and AblFL  was synthesized and cloned by GenScript into pETm41 . ResidueBuffer E: 50 mM Tris-HCl, 500 mM NaCl, 1 mM TCEP, pH 8.0Buffer F: 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, 1 mM TCEP, pH 8.0Buffer G: 50 mM Tris-HCl, 1 mM TCEP, 10% glycerol, pH 8.0Buffer H: 50 mM Tris-HCl, 1 M NaCl, 1 mM TCEP, pH 8.0All titrations were carried out using Nano ITC (TA Instruments) and analyzed via the NanoAnalyze software either using the independent fit model or competitive replacement model. The first injection of each experiment was discarded according to the software manual.For AurA, danusertib (Selleckchem #S1107) was reconstituted to 100 mM in 100% DMSO and were diluted to appropriate concentration to match final 5% DMSO (vol/vol) for each experiment. An ADP-analogue, AMPCP, was used for competitive replacement experiments to measure and fitting of the binding of danusertib to AurA. All proteins were dialyzed in 20 mM Tris-HCl, 200 mM NaCl, 10% (vol/vol) glycerol, 5 mM TCEP, pH 7.5. AMPCP was resuspended with the same buffer and was matched to pH 7.5. DMSO was added prior to each experiment to match 5% between titrant and titrand. Each injection was added in 2 μl increments with 180 s interval at a constant stirring speed of 300 rpm and at 25°C. Concentrations used for the experiments are noted below.20 μM AurA + 150 μM Mb120 μM AurA / 50 μM Danusertib + 384 μM Mb120 μM AurA / 150 μM Danusertib + 384 μM Mb120 μM AurA + 150 μM Mb220 μM AurA / 150 μM Danusertib + 150 μM Mb220 μM AurA + 90.9 μM Mb320 μM AurA / 150 μM Danusertib + 90.9 μM Mb3100 μM AurA + 1290 μM AMPCP20 μM AurA / 2000 μM AMPCP + 150 μM Danusertib40 μM AurA / 140 μM Mb1 + 1000 μM AMPCP20 μM AurA / 2000 μM AMPCP / 140 μM Mb1 + 150 μM Danusertib80 μM AurA / 200 μM Mb2 + 977.6 μM AMPCP20 μM AurA / 2000 μM AMPCP / 200 μM Mb2 + 150 μM Danusertib80 μM AurA / 100 μM Mb3 + 1200 μM AMPCP20 μM AurA / 2000 μM AMPCP / 100 μM Mb3 + 150 μM Danusertib8 μM AurA + 51.7 μM Mb68 μM AurA + 25 μM Danusertib + 51.7 μM Mb6For Abl, N-Myr peptide (Myr-GQQPGKVLGDQR), ordered from Genscript, was used for competitive replacement experiments to measure and fitting of the binding of asciminib to Abl. All proteins were dialyzed in 50 mM Tris-HCl, 500 mM NaCl, 1 mM TCEP, pH 8.0. Imatinib-mesylate (Sigma #SML-1027) and asciminib (MedKoo #206490) were reconstituted to 10 mM in 100% DMSO and were diluted to appropriate concentration for each experiment. N-Myr peptide was resuspended with the same buffer and was matched to pH 8.0. DMSO was added prior to each experiment to match 5% between titrant and titrand. Each injection was added in 1–1.5 μl increments with 180 s interval at a constant stirring speed of 300 rpm and at 25°C. Concentrations used for the experiments are noted below.15 μM AblKD + 100 μM Imatinib15 μM AblKD / 50 μM Asciminib + 100 μM Imatinib15 μM AblFL + 100 μM Imatinib15 μM AblFL / 50 μM Asciminib + 100 μM Imatinib15 μM AblFL / 150 μM Asciminib + 100 μM Imatinib30 μM AblKD + 400 μM N-Myr30 μM AblKD / 50 μM Imatinib + 400 μM N-Myr30 μM AblFL + 400 μM N-Myr30 μM AblFL / 50 μM Imatinib + 400 μM N-Myr25 μM AblKD / 300 μM N-Myr + 250 μM Asciminib25 μM AblKD / 50 μM Imatinib / 300 μM N-Myr + 250 μM Asciminib25.5 μM AblFL / 300 μM N-Myr + 250 μM Asciminib25.5 μM AblFL / 50 μM Imatinib / 300 μM N-Myr + 250 μM Asciminib30 μM AblKD + 2500 μM AMPPCP30 μM AblFL + 2500 μM AMPPCP50 of danusertib to AurA, ADP-Glo™ Max assay (Promega #V7001) was used. 20 nM AurA in the absence or presence of either saturating concentration of Mb1 or Mb2 or Mb3 was incubated with 3 mM Lats2 (ATLARRDSLQKPGLE), 0.6 mg/ml BSA and varying concentrations of danusertib with final 5% (vol/vol) of DMSO at 25 °C in 20 mM Tris-HCl, 200 mM NaCl, 10% (vol/vol) glycerol, 5 mM TCEP, pH 7.50. The bolded and underlined residue indicate site of phosphorylation. The reaction was initiated by adding 5 mM ATP, and the final samples were collected after 2 h for AurA-Mb1 complex, 10 h for apo AurA, and 20 h for AurA-Mb2 and AurA-Mb3 complexes. The amount of ADP in the samples were measured by following the manufacturer’s protocol and used to calculate the observed rate.To measure the ICYGEFKK), 0.6 mg/ml BSA, 20 mM MgCl2, 750 μM NADH, 6 mM PEP, 2.5 units of PK/LDH (Sigma #P0294). The bolded and underlined residue indicate site of phosphorylation. Oxidation of NADH at A340 was monitored using SpectraMAX by starting the assay with 1 mM ATP. Final volume of the assay was 100 μL. Observed rate (kobs) was calculated following Zorba et al.  in 50 mM Tris-HCl, 500 mM NaCl, 1 mM TCEP, pH 8.0 supplemented with 20 nM Abl kinase (AblKD or AblFL), 2 mM Srctide (EIAll-atom molecular dynamics simulations were conducted using OpenMM 7.6  and ‘MakFluoroMax-4 (Horiba Scientific) with temperature controller (water bath) was used to measure FRET between intrinsic tryptophan fluorescence and SKI. Either 10 nM Abl or 10 nM Abl + 200 nM asciminib were pre-incubated with varying concentrations of SKI for 40 min at 25 °C before measurements. An increase in the fluorescence was measured when the complex, specifically tryptophan, was excited at 295 nm to emit at 340 nm, which then excites SKI to emit at 460 nm . Both 5 The fluorescence intensity at 460 nm versus SKI concentration was fitted to quadratic equation below in GraphPad Prism to obtain apparent.ntration .2 with 2 μl reservoir of 0.1 M MES pH 6.5 + 0.2 M Ammonium sulfate + 4% (v/v) 1,3-propanediol + 15–18% PEG 8000. Streak seeding was used to obtain bigger crystals. Crystals were grown at 18 °C by hanging drop. The crystals were transferred to a drop of fresh reservoir for 30 seconds to remove excess of nucleotides from the crystal surface. Then, the crystals were transferred to a drop with reservoir with 1 mM danusertib for 16 h of soaking. For cryo-protection, the crystals were transferred into 17.5% PEG 400, 17.5% ethylene glycol, 15% reservoir, 50% water for a few seconds. Crystals of AurA in complex with Mb2 and danusertib were obtained by combining 0.5 μl of 300 μM (10 mg/mL) AurA + 315 μM (4 mg/mL) Mb2 + 1 mM danusertib with 0.5 μl of 0.1 M BIS-TRIS pH 5.5 + 0.2 M Ammonium acetate + 25% PEG3350. Crystals were grown at 18 °C by sitting drop. Crystals were harvested and subsequently flash frozen. Diffraction data for AurA-danusertib-Mb1 and AurA-danusertib-Mb2 were collected at 100 K Advanced Light Source  at beamlines BL821 and BL501, respectively, and were integrated with XIA2  AurA + 315 μM (4 mg/mL) Mb1 + 2 mM AMPPCP + 4 mM MgClith XIA2  or XDS  in 5% DMSO with 0.4 μl reservoir of 0.1 M Tris-HCl pH 8 + 1.75 M Ammonium sulfate + 2% (v/v) polypropylene glycol 400 (PPG 400). The final stock of complex was concentrated from 1 μM AblFL with ~1.2 μM SKI/asciminib after incubation at 4 °C for 6 h. Screening around this condition yielded crystals in a transparent diamond-shaped or plate-shaped crystals. Crystals were grown at 18 °C by sitting drop for a few days. The crystals were transferred to a drop of fresh reservoir containing 20% Xylitol with matching concentration of inhibitors in 5% DMSO for few seconds for cryo-protection. Single crystal X-ray diffraction data was collected at 100 K at Advanced Light Source (ALS) Berkeley (BL201). Data were integrated with XDS  as well Supplement 1