Carallia brachiata—a medicinal terrestrial plant from Rhizophoraceae. Furofuran lignans [(−)--buddlenol D (1) and (−)--buddlenol D (2)] isolated from the stem and bark inhibited nitric oxide : 9.25 ± 2.69 and 8.43 ± 1.20 micromolar for 1 and 2, respectively) and prostaglandin E2  productions in lipopolysaccharide-induced RAW264.7 cells. From western blotting, 1 and 2 suppressed LPS-induced inducible nitric oxide synthase and cyclooxygenase-2 expression in a dose-dependent manner (0.3–30 micromolar). Moreover, analysis of the mitogen-activated protein kinase (MAPK) signaling pathway showed decreased p38 phosphorylation levels in 1- and 2-treated cells, while phosphorylated ERK1/2 and JNK levels were unaffected. This discovery agreed with in silico studies which suggested 1 and 2 bound to the ATP-binding site in p38-alpha MAPK based on predicted binding affinity and intermolecular interaction docking. In summary, 7′′,8′′-buddlenol D epimers demonstrated anti-inflammatory activities via p38 MAPK inhibition and may be used as viable anti-inflammatory therapies.Excessive macrophage activation induces the release of high levels of inflammatory mediators which not only amplify chronic inflammation and degenerative diseases but also exacerbate fever and retard wound healing. To identify anti-inflammatory molecules, we examined  Additionally, non-infectious stimuli such as cell damage and tissue injury also trigger these responses and lead to local inflammation. Uncontrolled acute inflammation gradually destroys tissues and organs, diminishes their functions, and evolves into chronic inflammation and degenerative diseases such as osteoarthritis, rheumatoid arthritis, and even cancer2. When exposed to chemoattractants, tissue monocytes evolve to macrophages which engulf and destroy microbes in phagolysosomes using various hydrolytic enzymes and substances, including nitric oxide (NO). Moreover, NO is released into the extracellular fluid and functions as an inflammatory mediator which enhances phagocyte migration to injured or infected sites to amplify immune responses. However, excessive NO production limits recovery processes and destroys surrounding tissues4. Toll-like receptors (TLRs), especially TLR-4 which is a pathogen recognition receptor, and mitogen-activated protein kinase (MAPK) signaling pathways have important roles during inflammatory responses. Their deactivation mitigates acute sepsis, chronic inflammation, and degenerative diseases6. Eritoran, a structural lipopolysaccharide (LPS) lipid A mimic, and SB203580, a pyridinylimidazole derivative, both target TLR-4 and p38 MAPK, respectively. Additionally, curcumin and its analog L48H37 are effective in inhibiting TLR-48. Furthermore, p38 MAPK inhibition is beneficial for Alzheimer’s disease treatment9. Natural compounds such as icariin, apigenin, and astaxanthin also decrease amyloid-β-induced neurotoxicity via p38 MAPK inhibition9.Inflammation is a cascade event generated by innate immunity responses against microbial infectionCarallia brachiata (Lour.) Merr. (Rhizophoraceae) is one such natural source whose stem and bark are used for antipyretic remedies. Similarly, in Ayurveda  the stem bark is also used to treat oral ulcers and stomatitis10. In a previous study, ethyl acetate (EtOAc) and methanol (MeOH) bark extracts showed significant wound healing properties in incision and excision wound rat models11. Furthermore, MeOH and hydro-ethanolic leaf extracts exhibited analgesic, anti-inflammatory, and anti-diabetic activities in in vivo models13. Based on these traditional uses and pharmacological activities, the anti-inflammatory activity of this plant appears to underlie its mechanism of action.Natural products are vital sources of medicines and have been used extensively by different populations; in Thailand, traditional medicines have been used for centuries. Based on Thai traditional medicine, O-β-apiofuranosyl-(1 → 6)-β-glucopyranoside], and flavonoid glycosides such as apigenin-7-O-α-rhamnoside-(1 → 2)-β-glucopyranoside were isolated from leaves of C. brachiata16. Also, p-hydroxy benzoic acid and two proanthocyanidins [carallidin and mahuanin A], which possessed anti-radical scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals and inhibited xanthine oxidase, were isolated from bark17. To the best of our knowledge, no evidence exists in the literature to support the compounds contributing to the activities addressed in traditional uses of this plants, especially for anti-inflammatory activity which is linked antipyretic and wound healing properties. In this work, we identified compounds which exerted anti-inflammatory activities, examined their effects on p38 MAPK signaling pathway, and developed in silico models to identify ligand binding targets.In terms of chemical investigations, two pyrrolidine alkaloids [hygroline and (+)-pseudohygroline], a megastigmane diglycoside , together with (+)-7″R,8′′S:7″′R,8′′′S-hedyotisol A (3), (−)-syringaresinol (4), two modified stilbene dimers [(+)-diptoindonesin D (5) and (+)-parviflorol (6)], a proanthrocyanidin [(−)-mahuanin A (7)], 4-hydroxy-2-methoxyphenyl-6-O-syringoyl-β-D-glucopyranoside (8), and three benzoic acid derivatives  were identified by spectroscopic analysis and compared with the literature  values of 0.44 and 0.48, respectively, when triply developed in 20% acetone/dichloromethane (Me2CO/CH2Cl2) -C. brachiata stem and bark, the crude MeOH and its partitioned extracts were preliminary tested for NO inhibition in murine macrophages (RAW264.7). The EtOAc extract was selected for further isolation. All isolates were screened for NO inhibition at a final concentration of 30 μM. Only buddlenols D 1 and 2, which exhibited > 50% inhibition, underwent half maximal inhibitory concentrations (IC50) analysis. In pre-treated RAW264.7 cells with 1 and 2 prior to stimulation with 500 ng/mL LPS, 1 and 2 inhibited NO production in a concentration-dependent manner (0.3–30 µM) and exhibited no cytotoxicity  was used as a positive control showed percentage of NO inhibition at 78.69 ± 1.48%  production via the arachidonic acid pathway. Buddlenols D 1 and 2 were evaluated for PGE2 inhibition as a putative fever suppressant. Cell pretreatment with 1 and 2 inhibited LPS-induced PGE2 production in a concentration-dependent manner; IC50 values for 1 and 2 were 6.15 ± 0.39 and 5.70 ± 0.97 µM, respectively, whereas dexamethasone (10 µM) exhibited an inhibition percentage of 91.21 ± 1.17%  almost completely ablated iNOS and COX2 expression , and c-Jun N-terminal kinase (JNK) phosphorylation. Treatment with 500 ng/mL LPS significantly increased p-p38, p-ERK1/2, and p-JNK levels detected at 24-h incubation when compared with non-stimulated cells. Dexamethasone (10 µM) also significantly suppressed these levels when induced by LPS and suggested its anti-inflammatory activity may be related to the inhibition of p38, ERK1/2, and JNK phosphorylation. In cells pre-treated with buddlenols 1 and 2, p38 phosphorylation was significantly reduced when compared with LPS-treated cells. However, ERK1/2 and JNK phosphorylation levels were not significantly changed. Therefore, the effects of 1 and 2 on NO and PGE2 suppression may be due to inhibited p38 phosphorylation  site as their fitness scores for the allosteric pocket were much lower than BIRB-796, a p38-α non-ATP site inhibitor. For TLR-4, 1 and 2 exhibited approximately a 0.8-fold decrease in fitness score when compared with ZINC25778142, a TLR-4 inhibitor. Additionally, we investgatged protein–ligand intermolecular interactions in the ATP-binding site of p38-α MAPK and TLR4-MD2 (myeloid differentiation protien 2) interface using the most likely occurring conformation (highest fitness score) .The ADMET data Table  showed t1 and 2, adduct ions [M+Cl]− at m/z of 679.2166 and 679.2156 were observed by HR-ESI–MS, and calculated for their molecular formula, C33H40O13 of 1 and 2, respectively. From Heteronuclear Multiple Bond Correlation (HMBC) spectra, hydroxy protons at δH 7.05 and 7.15 of 1 were placed on C-4″  and C-4′ , respectively. Additionally, a C8″-O-C4 ether linkage of both substructures was suggested by carbon chemical shifts at C-8″ (δC 88.0) and C-4 (δC 135.9)18. From NMR analysis, HMBC correlations , and 13C-NMR signals of C-8″ (δC 89.4) and C-4 (δC 136.2) were also observed in 2. Relative configurations on C-8″ and C-7″ of 1 and 2 were guided by NOESY correlations. The Nuclear Overhauser Effect Spectroscopy (NOESY) spectrum of 1 showed a prominent correlation of δH 4.18 (H-8″) and δH 4.98 (H-7″) and a relatively weak correlation of δH 4.18 (H-8″)/δH 4.38 (OH-7″), while a strong correlation of δH 4.00 (H-8″) and δH 4.34 (OH-7″) and a relatively pale correlation of δH 4.00 (H-8″)/δH 4.96 (H-7″) were observed in NOESY spectrum of 2. Moreover, J7′′,8′′ of 1 and 2 were observed at 4 and 6.8 Hz, respectively, and corresponded with erythro- and threo- forms of an acyl glycerol moiety19. Thus, 7′′,8′′-erythro and 7′′,8′′-threo buddlenols D were deduced for 1 and 2, respectively, and supported chemical shift differences for 7″ and 8′′ positions (ΔδC8′′-C7′′) as suggested by Xiong et al.19; . From circular dichroism (CD) spectra of 1 and 2, positive Cotton effects were observed at 239 and 233 nm, respectively, indicating the 8″S configuration of both compounds19. Additionally, negative benzene 1Lb-band Cotton effects observed at 283 and 282 nm for 1 and 2, respectively, were consistent with 4  and revealed 7R,7′R,8S,8′S configurations in these molecules20. The absolute configuration of benzylic carbon (C-7″) bearing a hydroxy group in 1 and 2 was suggested by Cotton effects in Rh2(OCOCF3)4-induced CD experiments, based on the secondary alcohol bulkiness rule. E band signs (≈ 350 nm) were observed as negative and positive Δε for 1 and 2, respectively, suggesting the 7″R configuration for 1 and 7″S configuration for 221. Moreover, the assigned configurations of 1  and 2  were consistent with the electronic circular dichroism (ECD) spectrum calculated based on Density Functional Theory (DFT) 22. Later, the absolute configuration of two buddlenol D isomers  were thoroughly investigated in work by Xiong et al.19. Both 7′′R,8′′S- and 7′′S,8′′S-buddlenols D were isolated from Sinocalamus affinis (Poaceae) and Melodinus cochinchinesis (Apocynaceae) using reverse phase chromatography23. In our study these 7″,8″-buddlenol D epimers were separated by silica gel column chromatography eluted in 20% Me2CO/CH2Cl2. Buddlenol D and syringaresinol are furofuran lignans comprised of synapyl alcohol units, while hedyotisol A comes from coniferyl alcohol monomers. In our study, phenolic compounds  which possessed substructural guaiacyl and syringyl moieties, oligostilbenes , and a proanthocyanidin (7) demonstrated the influence of shikimate pathway on the biosynthesis of compounds stored in C. brachiata stem and bark. Although 7″,8″-buddlenol D epimers were isolated from the aforementioned plants, their biological activity and medicinal use were not previously addressed.Buddlenol D was first isolated from 2, a pyrogenic mediator, via suppressed COX2 expression by 1 and 2 supported the antipyretic properties of C. brachiata stem and bark for traditional use, and was putatively related to the finding that a higher core temperature correlated with higher PGE2 production24. Additionally, 1 and 2 significantly inhibited iNOS expression induced by LPS treatment. As LPS-induced iNOS and COX2 expression, these results suggested 1 and 2 exerted anti-inflammatory activities by suppressing these proinflammatory enzymes and reducing NO and PGE2 levels. MAPK signal transduction, including ERK1/2, JNK, and p38 MAPK, modulates inflammatory processes in response to stimuli via different receptors25. ERK1/2, JNK, and p38 MAPK phosphorylation activities are the major downstream signaling phenomena after TLR4 activation. Therefore, molecules regulating TLR4-mediated ERK1/2, JNK, and p38 MAPK phosphorylation may exhibit anti-inflammatory potential. From our data, we hypothesized the underlying mechanisms of 1 and 2 may occurred via modulated p38 MAPK phosphorylation. In contrast, dexamethasone, which is an anti-inflammatory drug and used as a positive control inhibited all ERK1/2, JNK, and p38MAPK phosphorylation26 and exerted potent anti-inflammatory activity by suppressing NO and PGE2 production and down-regulating iNOS and COX2 expression27.We also reported the anti-inflammatory activity of 7″,8″-buddlenol D epimers. The inhibition of PGE1 and 2 inhibited p38 MAPK phosphorylation, a specific p38 inhibitor SB203580 was used to identify anti-inflammatory activity. In RAW264.7 cells pre-treated with SB203580 prior to LPS stimulation, p38 MAPK phosphorylation suppression was clearly observed and resulted in NO inhibition  activities of 1 and 2 support the use of C. brachiata extracts for oral stomatitis and wound healing32 . Hence, molecular docking studies suggested that 1 and 2 could selectively impede p38-α MAPK at the ATP-binding site.Concerning the sharing features of ATP-binding pocket among kinases, we thus observed the binding capability toward other kinases involved in the focused signaling cascade. As listed in Table 1 and 2 might possess low GI absorption profile, based on in silico calculation 1 and 2 exhibited good metabolic profiles. Compounds 1 and 2 could be potential compounds for further structural development for better pharmacokinetic profiles and suitable drug delivery system. Moreover, after oral administration of 1 and 2, they might lose the syringyl unit during the digestion process and could be transformed to enterodiol derivatives by gut microbiome37. There was evidence that enterodiol exhibited p38 down regulation as one of the mechanisms inhibiting the growth of colorectal cancer cells38.Although, S-deoxydihydroglyparvin which is a sulfur-containing propanamide derivative isolated from Glycosmis parva (Rutaceae) leaves39. As furofuran lignans such as epimagnolin B from Magnolia fargesii (Magnoliaceae), (−)-sesamin-2,2′-diol from Isodon japonicus (Lamiaceae), and zanthpodocarpins A and B from Zanthoxylum podocarpum (Rutaceae) displayed anti-inflammatory activity via NO inhibition in cultured cells42, we suggest this compound class may exhibit anti-inflammatory activity. From our study, we propose 7″,8″-buddlenol D epimers function as p38 MAPK inhibitors.In our previous work, we reported a natural p38 MAPK inhibitor (+)-C. brachiata were collected in January 2018 from the botanical gardens of the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok. The samples were identified by a study author (C. Chansriniyom). The herbarium specimen (CC-CB-010118) was deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Chulalongkorn University. In addition, the handling and notification of stems and barks of C. brachiata were carried out in accordance with the Plant Variety Protection Act B.E. 2542 (1999): Section 53, the Kingdom of Thailand.Stems and barks from C. brachiata stems and barks (4.0 kg) were successively macerated in MeOH (3 × 15 L) to yield a 203.80 g MeOH extract which was partitioned with hexane to generate a hexane extract (7.34 g). The remaining organic layer was added to water and further partitioned with EtOAc and n-butanol (n-BuOH) to obtain EtOAc (27.39 g), n-BuOH (50.39 g), and aqueous (113.89 g) extracts.Dried and coarsely powdered ® XS 420, Advion Inc., NY, USA) and was eluted in a gradient solvent of MeOH, EtOAc and hexane at an initial ratio of 0:40:60 (respectively), then progressed to 30:70:0 (respectively), and terminated in 100% MeOH. Based on chromatographic patterns fractions were combined to generate seven fractions (CBE1–CBE7).The EtOAc extract (27.39 g) underwent silica gel column chromatography (Flash column chromatography (Flash CC), PuriFlash2Cl2 system ]. CBE7-3 (874 mg) was separated into 48 fractions using Medium Pressure Liquid Chromatography  . CBE7-3-12 (178.2 mg) was purified using MPLC  to obtain 1 (6 mg) and 2 (2.4 mg). CBE7-3-20 (88.9 mg) was separated by MPLC  to afford 3 (6 mg). CBE7-4 (466 mg) was purified using two MPLC steps  to achieve 8 (6.3 mg).CBE7 (6.58 g) was separated into seven sub-fractions using Flash CC +. HR-ESI–MS m/z: 679.2166 [M+Cl]−, calcd. for C33H40O13Cl 679.2163. [α]20D: − 12.96° . CD nm (MeCN): 204.8 (Δε − 13.88), 208.7 (Δε − 1.50), 213.1 (Δε − 5.35), 217.9 (Δε + 3.23), 225.7 (Δε − 4.14), 234.3 (Δε + 2.06), 239.0 (Δε + 0.50), 244.6 (Δε − 0.73), 283.3 (Δε − 0.09). Rh2(OCOCF3)4-induced CD nm (CH2Cl2): 355.4 (Δε − 0.83) -4′,4′′-Dihydroxy-3,3′,3′′,5,5′,5′′-hexamethoxy-7,9′:7′,9-diepoxy-4,8′′-oxy-8,8′-sesquineolignan-7′′,9′′-diol : white solid. 1H-NMR  δ: 7.15 , 7.06 , 6.76 , 6.74 , 6.68 , 4.96 , 4.73 , 4.68 , 4.34 , 4.25 , 4.00 , 3.90 , 3.87 , 3.82 , 3.79 , 3.67 , 3.36 , 3.11 ; 13C-NMR  δ: 153.9 , 148.8 , 148.4 , 139.3 (C-1), 136.3 (C-4′), 136.2 (C-4), 136.1 (C-4″), 133.2 (C-1′), 132.8 (C-1″), 105.4 , 104.5 , 104.0 , 89.4 (C-8″), 86.8 (C-7′), 86.6 (C-7), 74.1 (C-7″), 72.6 , 61.6 (C-9″), 56.7 , 55.4 ; IR (ATR) cm−1: 3466 (broad), 2939, 2844, 1610, 1594, 1520, 1459, 1216, 1116; UV λmax (MeOH) nm (log ε): 206 (4.64), 236 sh (3.93), 272 (3.24). ESI–MS m/z: 667 [M+Na]+. HR-ESI–MS m/z: 679.2156 [M+Cl]−, calcd. for C33H40O13Cl 679.2163. [α]20D: − 9.78° . CD nm (MeCN): 203.9 (Δε − 12.58), 207.7 (Δε + 3.04), 210.1 (Δε + 1.19), 214.8 (Δε + 6.30), 219.0 (Δε − 0.24), 221.4 (Δε + 0.42), 225.5 (Δε − 0.98), 233.1 (Δε + 0.61), 237.8 (Δε − 0.62), 282.2 (Δε − 0.04). Rh2(OCOCF3)4-induced CD nm (CH2Cl2): 347.2 (Δε + 0.81) -.Mouse macrophage cells RAW264.7  were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum in 5% CO5 cells/mL) in 100 µL were seeded in a 96‑well plate and incubated for 24 h at 37 °C. After this, cells were treated with different compound concentrations and incubated for 24 h. Then, medium containing compound was replaced by phenol red-free medium containing LPS   and the cells were further incubated for 24 h. NO levels were determined by collecting 100 µL supernatants and mixing with 100 µL modified Griess reagent (Sigma-Aldrich). Reaction mixtures were incubated for 15 min at room temperature and absorbance measured at 560 nm using a microplate reader. NO levels were quantified by interpolation using a nitrite standard curve. Cells treated with dexamethasone (10 µM) were used as a positive control group.Cells (5 × 105 cells/mL) in 100 µL were seeded in a 96‑well plate and incubated for 24 h at 37 °C. Then, cells were treated with different compound concentrations and incubated for 24 h. After that, medium containing compound was replaced by medium containing LPS  and the cells were incubated for another 24 h. Supernatants were collected and subjected to enzyme-linked immunosorbent assay for PGE2  following manufacturer’s instructions.Cells , anti-COX2 (CST#2282), anti-phospho-ERK (CST#4376), anti-ERK (CST#4695), anti-phospho-JNK (CST#4668), anti-JNK (CST#9252), anti-phospho-p38 (CST#4511), anti-p38 (CST#9212), and followed by incubation with a horse radish peroxidase-conjugated secondary antibody. Protein bands were detected using a chemiluminescent substrate and images acquired using ImageQuant LAS 4000 . Relative protein band intensity was normalized to β-actin or GAPDH expression using ImageJ. SB203580  is a specific p38-α inhibitor and was used as a positive control for p38 phosphorylation inhibition.44, 3ZSH33, 1KV245, 4QTB46, 1PME47, 2NO348, 3NPC49, 4W4Y50, 4KIK51, respectively. In addition to protein preparation, the 3-dimensional structures of 1 and 2 were manually constructed using the Gaussian 09 program and optimized using the HF/6-31d basis set implemented in the program52. For docking studies, the Gold docking program was used, and protocols were as follows: (1) the ligand binding site was defined as 6 Å for sphere docking and (2) ChemScore was used for the scoring function. Binding between proteins and compounds was visualized in Accelrys Discovery Studio 2.5 (Accelrys Inc.).Protein–ligand docking was conducted for eight targeted proteins under nine systems-TLR4, p38-α MAPK (ATP-binding site), p38-α MAPK (non-ATP-binding site), ERK1, ERK2, JNK1, JNK2, JNK3 and IκB kinase β using crystallized structures (retrieved from RCSB Protein Data Bank), listed as follows: 2Z6534 and the docked pose was then observed its orientation and intermolecular interactions with the key reported residues including D209, S211, and D234. Our docking protocols were validated for all systems 53 while their toxicity profiles were predicted using Protox-II platform (https://tox-new.charite.de/protox_II/)54 and the DataWarrior software package55.Pharmacokinetic properties  of compounds p < 0.05.All data were expressed as the mean ± standard error of the mean from at least three independent experiments. Differences among groups were evaluated using one-way analysis of variance, followed by post-hoc tests with Bonferroni correction. Statistical significance was accepted at C. brachiata stem and bark. Their action mechanisms were mediated by inhibited p38 MAPK phosphorylation which attenuated iNOS and COX2 expression and inhibited NO and PGE2 production in LPS-induced RAW264.7 macrophages. These findings support the use of C. brachiata for antipyretic, oral stomatitis, and wound healing purposes. Therefore, 7″,8″-buddlenol D epimers can be used as biological markers for C. brachiata extract.For the first time, we characterized the anti-inflammatory activities of 7″,8″-buddlenol D epimers from Supplementary Information.


We prove that soft ω-regular open sets of a soft topological space form a soft topology. Also, we prove that soft semi ω-regularity and soft almost ω-regularity are independent notions. In addition, we reveal the relationships between soft topology and classical (parametric) topology. Finally, we characterize soft nearly Lindelöfness and improve several results related to soft nearly Lindelöfness using the concept of soft ω-regular open sets.In this paper, we define the notion of “soft  Soft regular open; Soft semi regular; Soft almost regular; Soft nearly Lindelöf; Soft generated soft topological space; Soft induced topological spaces Accordingly, it is not possible to deal with these problems by traditional methods. For more than thirty years, fuzzy set theory Shabir and Naz Closure and interior operators were used to give rise to several different new classes of sets, while a few others are the so-called regular sets. Researchers have discovered applications for these regular sets not only in mathematics but even in a variety of fields outside of mathematics ω-regular open sets via soft topological spaces, to characterize soft nearly Lindelöfness, to improve several results related to soft nearly Lindelöfness using the concept of soft ω-regular open sets, and to open the door to redefine and investigate some of the soft topological concepts such as soft compactness, soft menger, soft separation axioms, and soft continuity, via soft ω-regular open sets.The targets of this paper are to scrutinize the behaviors of soft The arrangement of this article is as follows:ω-regular open sets. We investigate the properties of these soft sets and point out their relationships with some famous generalized soft open sets. We also show the relationships between soft ω-regular open sets in soft topology and their general topological counterparts.In Section ω-regular and soft almost ω-regular. We investigate their properties and point out their relationships with some famous soft separation axioms. We also show the relationships between each of them in soft topology and their general topological counterparts.In Section ω-regular open sets.In Section In Section Definition 1.1Z be a universal set and E be a set of parameters. A soft set over Z relative to E is a function Z relative to E will be denoted by Definition 1.2Let K will be denoted by (a) K will be denoted by (b) K will be denoted by (c) Definition 1.3For any (a) (b) (c) Definition 1.4M is said to be a soft subset of N, denoted by (a) M and N is denoted by (b) the soft union of M and N is denoted by (c) the soft intersection of M and N is denoted by (d) the difference of Definition 1.5(a) the soft union of these soft sets is the soft set denoted by (b) the soft intersection of these soft sets is the soft set denoted by Definition 1.6M (Definition 1.7δ ⊆ Z relative to E if(a) δ is closed under arbitrary soft union,(b) δ is closed under finite soft intersection.(c) δ is a soft topology on Z relative to E, then the triplet δ are called soft open sets in If U in U in K in K in From now on, topological space and soft topological space will be denoted by TS and STS, respectively. Let Definition 1.8U of a TS The family of all soft regular open sets (resp. regular closed sets) of a TS Definition 1.9W of a TS ω-regular open set in ω-regular open set in ω-regular closed set.ω-regular open sets (resp. ω-regular closed sets) of a TS The family of all Definition 1.10A TS (a) (b) ω-regular if for every (c) ω-regular if for every (d) Definition 1.11S of a STS The collection of all soft regular open sets (resp. soft regular closed sets) of a STS Definition 1.12A STS (a) (b) (c) Definition 1.13(a) K is said to be soft nearly Lindelöf relative to K from (b) Theorem 1.14For any STSand any,is a topology on Z. This topology is denoted by.Theorem 1.15Letbe a TS and E be a set of parameters. Then(a)is a soft topology on Z relative to E. This soft topology is denoted by.(b)is a soft topology on Z relative to E. This soft topology will be denoted by.It is not difficult to check that the two types of soft topology in Theorem 1.16For any collection of TSs,is a soft topology on Z relative to E. This soft topology will be denoted by.Theorem 1.17For any STS,is a soft topology on Z relative to E. This soft topology is denoted by.A non-empty subset 2ω-regular open sets. We investigate the properties of these soft sets and point out their relationships with some famous generalized soft open sets. We also show the relationships between soft ω-regular open sets in soft topology and their general topological counterparts.In this section, we define soft Definition 2.1S of a STS ω-regular open set in ω-regular open set in ω-regular closed set.A soft set ω-regular open sets (resp. soft ω-regular closed sets) of a STS The family of all soft Theorem 2.2For any STS,.ProofStraightforward. □Theorem 2.3For any STS,.ProofLet The following example shows that the inclusion in Example 2.4ρ be the usual topology on Z, and Let Theorem 2.5For any STS,.ProofLet Inclusion in Example 2.6Let Theorem 2.7For any STS,is a STS.Proof(1) We have (2) Let Since (3) Let Since Theorem 2.8Letbe a STS and. Thenif and only if for every, there existsandsuch thatand.ProofNecessity. Suppose that Sufficiency. Suppose that for every Proposition 2.9Letbe a STS such that for some,for every. Then(a) For each,.(b) For each,.(c) For each,.Proof(a) Let (b) Let But (c) Let Theorem 2.10Letbe a STS such that for some,for every. Then for every,.ProofLet Hence, Theorem 2.11Letbe a STS such thatfor allandand let. Thenif and only iffor every.ProofNecessity. Suppose that Sufficiency. Suppose that Corollary 2.12Letbe a TS and E be any set of parameters. Let. Thenif and only if.Theorem 2.13Letbe a STS such that for some,for every. Then for every,.ProofZ. Therefore, Let Theorem 2.14Letbe a STS such thatfor alland, and let. If, thenfor every.ProofSuppose that Question 2.15Let The following theorem gives a partial answer for Theorem 2.16Letbe a TS and E be any set of parameters. Let. Thenif and only if.ProofNecessity. Suppose that Sufficiency. Let Theorem 2.17Letbe a collection of TSs. Thenif and only iffor all.ProofNecessity. Suppose that Sufficiency. Suppose that Corollary 2.18Letbe a TS and E be any set of parameters. Let. Thenif and only iffor every.3ω-regular and soft almost ω-regular. We investigate their properties and point out their relationships with some famous soft separation axioms. We also show the relationships between each of them in soft topology and their general topological counterparts.In this section, we define two new classes of soft separation axioms: soft semi Definition 3.1ω-regular if for each A STS Theorem 3.2Every soft semi regular STS is soft semi ω-regular.ProofFollows from the definitions and ω-regularity does not imply soft semi regularity in general:Soft semi Example 3.3Let ω-regular. On the other hand, since Theorem 3.4Letbe a soft semi regular STS such that for some,for every. Thenis semi regular.ProofLet Corollary 3.5Ifis a soft semi regular STS such thatfor alland, thenis semi regular for every.Question 3.6Let The following theorem gives a partial answer for Theorem 3.7Letbe a TS and E be any set of parameters. Thenis soft semi regular if and only ifis semi regular.ProofNecessity. Suppose that Sufficiency. Suppose that Theorem 3.8Letbe a collection of TSs. Thenis soft semi regular if and only ifis semi regular for all.ProofNecessity. Suppose that Sufficiency. Suppose that Corollary 3.9Letbe a TS and E be any set of parameters. Thenis soft semi regular if and only ifis semi regular.Theorem 3.10Letbe a soft semi ω-regular STS such that for some,for every. Thenis semi ω-regular.Proofω-regular, then there exists ω-regular. □Let Corollary 3.11Ifis a soft semi ω-regular STS such thatfor alland, thenis semi ω-regular for every.Question 3.12ω-regular for all ω-regular?Let The following theorem gives a partial answer for Theorem 3.13Letbe a TS and E be any set of parameters. Thenis soft semi ω-regular if and only ifis semi ω-regular.ProofNecessity. Suppose that ω-regular. Choose ω-regular. But ω-regular.Sufficiency. Suppose that ω-regular. Let ω-regular, then there exists ω-regular. □Theorem 3.14Letbe a collection of TSs. Thenis soft semi ω-regular if and only ifis semi ω-regular for all.ProofNecessity. Suppose that ω-regular and let ω-regular, then there exists ω-regular.Sufficiency. Suppose ω-regular for all ω-regular, then there exists ω-regular. □Corollary 3.15Letbe a TS and E be any set of parameters. Thenis soft semi ω-regular if and only ifis semi ω-regular.Definition 3.16ω-regular if for each A STS Theorem 3.17Every soft almost ω-regular STS is soft almost regular.ProofFollows from the definitions and ω-regularity and soft almost ω-regularity are independent concepts:The following two examples will show that the converse of Example 3.18ω-regular.Let Example 3.19ω-regular. On the other hand, since ω-regular.Let Theorem 3.20Every soft semi ω-regular soft almost ω-regular is soft regular.Proofω-regular and soft almost ω-regular. Let ω-regular, then there exists ω-regular, then there exists Let Theorem 3.21Letbe a TS and E be any set of parameters. Thenis soft almost regular if and only ifis almost regular.ProofNecessity. Suppose that Sufficiency. Suppose that Theorem 3.22Letbe a TS and E be any set of parameters. Thenis soft almost ω-regular if and only ifis almost ω-regular.ProofNecessity. Suppose that ω-regular. Let ω-regular and ω-regular.Sufficiency. Suppose that ω-regular. Let ω-regular and ω-regular. □Theorem 3.23Letbe a collection of TSs. Thenis soft almost regular if and only ifis almost regular for all.ProofNecessity. Suppose that Sufficiency. Suppose that Corollary 3.24Letbe a TS and E be any set of parameters. Thenis soft almost regular if and only ifis almost regular.Theorem 3.25Letbe a collection of TSs. Thenis soft almost ω-regular if and only ifis almost ω-regular for all.ProofNecessity. Suppose that ω-regular and let ω-regular, then there exists ω-regular.Sufficiency. Suppose that ω-regular for all ω-regular and ω-regular. □Corollary 3.26Letbe a TS and E be any set of parameters. Thenis soft almost ω-regular if and only ifis almost ω-regular.ω-regularity:We end this section with a characterization of soft almost Theorem 3.27A STSis soft almost ω-regular if and only if for eachand eachsuch that, there existssuch that.ProofNecessity. Suppose that ω-regular. Let Sufficiency. Obvious. □4ω-regular open sets.In this section, we characterize soft nearly Lindelöfness and improve several soft nearly Lindelöfness-related results by employing the concept of soft We start with the following characterization of soft nearly Lindelöf STSs:Theorem 4.1For a STS,the following are equivalent:(a)is soft nearly Lindelöf.(b) Every soft cover offromhas a countable subcover.(c) For everywith, there exists a countable subfamilysuch that.(d) For everywith the countable soft intersection property,.Proof(a) ⟹ (b): Let H is countable and Since (b) ⟹ (c): Let (c) ⟹ (d): Suppose to the contrary that there exists (d) ⟹ (a): Let Theorem 4.2Letbe soft nearly Lindelöf STS. Then for each, K is soft nearly Lindelöf relative to.ProofLet K is soft nearly Lindelöf relative to then there exists a countable subfamily The following corollary is an immediate consequence of Corollary 4.3Letbe soft nearly Lindelöf space. Then for each, K is soft nearly Lindelöf relative to.Theorem 4.4Every soft semi ω-regular soft nearly Lindelöf STS is soft Lindelöf.Proofω-regular and soft nearly Lindelöf. Let δ. For each ω-regular, then for each Let Corollary 4.5Every soft semi regular soft nearly Lindelöf STS is soft Lindelöf.Corollary 4.6Every soft regular soft nearly Lindelöf STS is soft Lindelöf.Theorem 4.7Letbe soft almost regular and soft nearly Lindelöf space. Then for everysuch that, there existsuch that,, and.ProofSince Theorem 4.8Letbe soft almost ω-regular and soft nearly Lindelöf space. Then for everysuch that, there existsuch that,, and.Proofω-regular, for each Since 5ω-regular open sets have been introduced. It is proved that the collection of soft ω-regular open sets forms a soft topology that lies strictly between the classes of soft regular open sets and soft ω-open sets. Soft semi ω-regularity and soft almost ω-regularity have been also introduced as two new independent variants of soft regularity via soft ω-regular open sets. In addition, the relationships between soft topology and classical (parametric) topology have been revealed. Finally, nearly Lindelöfness has been characterized, and several results related to soft nearly Lindelöfness have been improved. In future studies, the following topics could be considered: 1) define new classes of soft functions using soft ω-regular open sets; 2) investigate the behavior of soft semi ω-regular and soft almost ω-regular STSs in the context of product STSs; and 3) characterize some types of soft Menger spaces using soft ω-regular open sets.Soft Samer Al Ghour: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.No data was used for the research described in the article.The authors declare no conflict of interest.No additional information is available for this paper.


In the extended structure of the title molecular salt, the cations and anions are linked by N—H⋯O hydrogen bonds to generate [010] chains. 6H9N2+·C7H7O3S−, the cations and anions are linked by N—H⋯O hydrogen bonds to generate [010] chains.In the extended structure of the title molecular salt, C The title salt was obtained by an aqueous reaction of the aromatic diamine with 4-methylbenzene-1-sulfonic acid in a 2:1 molar ratio, unlike 2, which was crystallized from a 1:2 reaction.In this report, we describe the crystal structure of the title salt, 1 consists of one 2-aminoanilinium cation and one 4-methylbenzene-1-sulfonate anion  was dissolved in aqueous ethanol (25–30 ml). Into this, an aqueous solution of 4-methylbenzene-1-sulfonic acid  was added. The clear reaction mixture thus obtained was left aside for crystallization. After a few days crystals of Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620002308/hb4337sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2414314620002308/hb4337Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620002308/hb4337Isup3.cmlSupporting information file. DOI: 1985013CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The entire afforded derivatives showed varied inhibition profiles ranging between 0.70 ± 0.01 and 30.80 ± 0.80 μM (against α-amylase) in comparison to standard acarbose (12.80 ± 0.10 μM). Similarly, synthetics analogs also displayed a varied range of α-glucosidase activity ranging from 0.80 ± 0.01 μM to IC50 = 29.70 ± 0.40 μM (against α-glucosidase) as compared to standard acarbose (IC50 = 12.90 ± 0.10 μM). Among synthesized analogs, compound 22 showed excellent potency due to the presence of di-hydroxy substitutions at the 2,3-position of the aryl ring. For all analogs, the structure–activity relationship was carried out based on the pattern of substitutions around the aryl ring, and further, the potent analogs were subjected to a molecular docking study to analyze how active residues of targeted enzymes interact with active parts of newly prepared analogs. The result obtained shows that these compounds furnish several key interactions with enzyme active sites and, hence, enhanced their enzymatic activities.Twenty-five analogs were synthesized based on 1,3,4-thiadiazole-fused--thiadiazole incorporating 1,4-benzodioxine moiety  A high concentration of glucose in plasma results in diabetes, which is a major metabolic problem . DiabeteBlood glucose levels over normal or hyperglycemia are indicated by impaired glucose tolerance (IGT) or impaired fasting blood glucose levels (IFG). In contrast to IFG, which occurs when blood glucose levels often exceed normal blood glucose concentrations above 7 mmol/L, IGT is characterized by blood glucose levels that are greater than 11 mmol/L 2 hours after a 75-g oral glucose load. In recent investigations of diabetes as a cause of male factor infertility, IGF and IGT were employed as diagnostic criteria. They are independent markers for diagnosis of diabetic mellitus (DM). These studies found that diabetes mellitus affects sperm motility, sperm counts, semen volume, and normal sperm morphology in addition to impairing erectile and ejaculatory function . There wIt was reported that numerous marketed drugs hold heteroarenes as the core center in their structure . Among hOne of the most difficult areas of drug development, particularly for oral-drug delivery systems, continues to be the enhancement of drug solubility and, consequently, its oral bioavailability. The solubility of poorly water-soluble medications can be improved using a variety of techniques that have been published in the literature. The methods are chosen based on a number of factors, including the qualities of the medicine under consideration, the kind of excipients to be chosen, and the kind of dosage form intended. Poor solubility and a sluggish rate of drug dissolution in aqueous gastrointestinal fluids are common causes of insufficient bioavailability for poorly water-soluble drugs. Especially for class II (low solubility and high permeability) drugs, increase in the drug’s solubility and rate of dissolution in gastrointestinal fluids may enhance bioavailability. Increasing solubility also enhances the bioavailability of Biopharmaceutical Classification System (BCS) class II medicines because, rather than absorption, the rate-limiting processes for BCS class II drugs are solubility in the stomach fluid and drug release from the dose form .α-glucosidase and α-amylase inhibition properties possessed by thiadiazole analogs  was reacted with thiosemicarbazide  in methanol (10 ml) under the catalytic action of sodium acetate (0.8 mmol) to afford 1,4-benzodioxine-based thiosemicarbazone as the substrate (II). This intermediate was cleaned with hexane and deionized water to eliminate all impurities. Substrate  was subjected to an iodine-mediated (0.6 mmol) cyclization in 1,4-dioxane (10 ml) in the presence of potassium carbonate (0.8 mmol), and the resulting mixture was stirred for 12–14 h under reflux to obtain 1,4-benzodioxine-based 1,3,4-thiadiazole-2-amine as an intermediate (III). In the last step, substrate  further undergoes [3+2] iodine-mediated (0.6 mmol) oxidative cyclization with different phenylisothiocyanates  in chloroform (10 ml) and potassium carbonate (0.8 mmol), and the resulting mixture was put on reflux for 16 h. It was reacted with a 5% sodium thiosulfate solution after being cooled to 25°C, followed by extraction with DCM (10 ml) to obtain targeted 1,4-benzodioxin-based 1,3,4-thiadiazole-fused--thiadiazole analogs (1–25) in an appropriate yield. The reaction progress was checked by employing a TLC plate. Different spectroscopic tools, including HREIMS and NMR, were employed to find the exact structure of the afforded analogs  inhibitory profiles for α-amylase and α-glucosidase. The entire analogs illustrated remarkable potency against α-amylase and α-glucosidase having IC50 values ranging from 0.70 ± 0.01 to 30.80 ± 0.80 μM (α-amylase) and 0.80 ± 0.01 to 29.70 ± 0.40 μM (α-glucosidase) when compared to standard acarbose drugs. It was suggested by SAR studies that each part of the synthesized analogs such as benzodioxine ring A, 1,3,4-thiadiazole-fused--thiadiazole and aryl ring B actively participated in the activity, and any variation around ring B resulted to different activity  bearing di-hydroxy substitutions at the 2,3-position of ring B showed excellent α-amylase activity when compared to other analogs 2 (IC50 = 3.40 ± 0.10 μM), 3 (IC50 = 4.40 ± 0.10 μM), 5 (IC50 = 1.10 ± 0.10 μM), 6 (IC50 = 5.20 ± 0.10 μM),7 (IC50 = 9.20 ± 0.20 μM), 12 (IC50 = 8.20 ± 0.20 μM), 18 (IC50 = 3.20 ± 0.10 μM), and analog 8  under the positive control of standard acarbose (IC50 = 12.80 ± 0.10 μM). This enhanced activity of analog 22 showed the presence of attached di-hydroxy groups at the 3,4-position, which could enhance the inhibitory potentials through active participation in conventional hydrogen bonding with enzyme active residues. Moreover, analog 8 bearing tri-hydroxy substitutions at the 2,4,6-position of ring B was found to be second most potent and displayed enhanced activity than di-hydroxy substitutions bearing analogs 2, 3, 5, and 6, indicating that any variation in the position of substitution around ring B may result to a different activity. In addition, analogs 2, 3, and 5 bearing di-hydroxy substitution at phenyl ring B displayed superior activity than analogs 7 and 12 bearing only mono-hydroxy substitution. This enhanced activity of analogs 2, 3, and 5 was due to greater numbers of the attached hydroxy groups that might have more tendencies to interact with the active site of the enzyme. However, by comparing structurally similar analogs 7, 18, and 12 having a mono-hydroxy substitution at meta-, ortho-, and para-position of the phenyl ring, among these analogs, 18 was more potent than its counterparts 7 and 12. This difference in activity shows that alteration in the position of substituted around phenyl ring B greatly affects the activity , 24 (IC50 = 5.30 ± 0.10 μM), and 25 (IC50 = 2.10 ± 0.10 μM), the analogs 23 and 25 bearing the –F moiety at the ortho- and para-position of the phenyl ring showed superior potency when compared to its counterpart 24 that bears the –F moiety at meta-position. The diverse potency of these analogs was due to different attachment of the –F moiety around aryl ring B , 3 , and 5 . This superior activity of analog 8 was due to the greater number/s of attached hydroxyl moieties at the 2,4,6-position of phenyl ring B  holding methyl substitution at the para-position of ring B displayed superior potency in comparison to analogs 15 (IC50 = 17.20 ± 0.30 μM) and 19 (IC50 = 18.40 ± 0.40 μM) that also hold methyl groups at the ortho- and meta-position of phenyl ring B. This difference in potency of these analogs was due to the different positions of the attached electron donating –CH3 groups around ring B , Ile235 (pi–sigma and H-bonding), Leu162 and Ala198 (pi–pi T-shaped), His201 and Trp59 (pi–pi stacking), and numerous van der Waals interactions, when evaluated against α-amylase as shown in The molecular docking had been conducted on α-amylase and α-glucosidase enzymes for most active compounds 8 bearing tri-hydroxyl groups at the 2,4,6-position of the aryl ring which can enhance its inhibitory potentials, this compound 8 when evaluated against α-amylase enzyme showed somewhat fewer interactions than compound 22, and, hence, its inhibitory potentials were found less than those of compound 22, which showed better binding interactions. Against α-amylase, this compound 8 revealed several interactions such as Lys200 (CHB), Tyr151 (pi–pi stacking), His201 (pi–sulfur & pi–cation), Ile235 , Ala198 (pi–pi T-shaped), Leu162 , and van der Waals interactions , Asp300 and Asp197 (pi–anion), Gly104 (carbon–hydrogen bond), Trp59 (pi–sulfur & pi–pi stacking), and van der Waals interactions  were conducted on a Finnigan MAT-311A instrument (Germany). The chromatogram was shown using UV light from Schimazdu, Germany, with a wavelength of 254/365.All biological and chemical components were bought from Merck KGaA and Sigma-Aldrich Chemical Corp., both of St. Louis, Missouri, United States . Using a Silica Gel 60 F254 TLC plate, thin-layer chromatography (TLC) was used to assess the purity of all intermediate and necessary chemicals . A Stuart Melting Point Apparatus SMP30 was used to measure melting points . The I  were stirred together in 10 ml of methanol followed by addition of a formyl group containing benzodioxine I to it. A rotary evaporator was used to evaporate the solvent after the reaction mixture was agitated at 25°C for 3 h. The desired benzodioxine-based thiosemicarbazone II was produced in excellent yield after the residue was triturated with ethyl acetate/diethyl ether (5:95).Thiosemicarbazide , sodium acetate (0.8 mmol), and benzodioxine II  in 1,4-dioxane (10 ml), potassium carbonate (0.8 mmol) and iodine (0.6 mmol) were successively added. In a nitrogen environment, the reaction mixture was heated to a reflux temperature until the formation of a new entity was complete . It was then brought to room temperature, treated with 20 ml of 5% Na2S2O3, and extracted using EtOAc (3 × 15 ml). Over anhydrous sodium sulfate, the mixed organic layer was dried and concentrated. In order to produce 2-amino-1,3,4-thiadiazole substrate III in a high yield, the supplied residue was purified using silica gel column chromatography with petroleum ether and EtOAc combination as the eluent.Further dissolving the intermediate (1–25) in an appropriate yield. The structures of all the newly discovered analogs were confirmed using spectroscopic techniques, including NMR and HREI-MS.Finally, 2-amino-1,3,4-thiadiazole-based substrate  further undergoes [3+2] oxidative cyclization on stirring overnight with different substituted phenyl isothiocyanates  in the presence of molecular iodine (0.6 mmol) and potassium carbonates (0.8 mmol) in chloroform (10 ml) as a solvent to access the targeted 1,3,4-thiadiazole-fused--thiadiazole-based benzodioxine analogs Utilizing Discovery Studio Visualizer’s (DSV) MGL tool 1.5.7 and AutoDock Vina, a molecular docking study was carried out . In this(1–25) of thiadiazole-fused--thiadiazole-incorporating benzodioxine moiety. The literature-recognized procedure was used to evaluate the synthesized analogs’ ability to inhibit α-amylase and α-glucosidase. When compared to standard acarbose (IC50 = 12.80 ± 0.10 μM), (IC50 = 12.90 ± 0.10 μM), all analogs demonstrated a diverse range of efficacy with IC50 values ranging from 0.70 ± 0.01 to 30.80 ± 0.80 μM (against α-amylase) and 0.80 ± 0.01 to 29.70 ± 0.40 μM (α-glucosidase). The structure–activity relationship was carried out for all analogs based on substitution pattern around the aryl ring, and it was concluded from SAR studies that analogs holding hydroxyl moieties around the aryl ring enhanced the enzymatic activities against both these targeted enzymes via the formation of a strong conventional hydrogen bond with enzyme active sites. Additionally, a molecular docking study was performed on the potent analogs to determine the best way for them to interact with the active residues of the intended enzymes. The results revealed that these compounds provide a number of important interactions with the enzyme active site, increasing the enzymes’ enzymatic activities. Additionally, a number of spectroscopic techniques, including HREI-MS, 13C-NMR, and 1H-NMR, were employed to investigate the precise structure of the synthesized analogs.In conclusion, we have designed and then synthesized twenty-five analogs


D ∼0.054 μM. Z0933M occupies a hydrophobic hotspot (P1) – encompassing an aromatic cage of two phenylalanines (F101 and F139) – alongside C-terminal extension of Skp1 and, thus, hampers its ability to interact with F-box proteins, a prerequisite step to constitute intact and active SCF E3 ligase(s) complexes. In cellulo, Z0933M disrupted SCF E3 ligase(s) functioning, recapitulated previously reported effects of Skp1-reduced activity, and elicited cell death by a p53-dependent mechanism. We propose Z0933M as valuable tool for future efforts toward probing Skp1 cancer biology, with implications for cancer therapy.Skp1 overexpression promotes tumor growth, whereas reduced Skp1 activity is also linked with genomic instability and neoplastic transformation. This highlights the need to gain better understanding of Skp1 biology in cancer settings. To this context, potent and cellularly active small-molecule Skp1 inhibitors may be of great value. Using a hypothesis-driven, structure-guided approach, we herein identify Z0933M as a potent Skp1 inhibitor with K  •Z0933M manifests strong binding with Skp1 and inhibits Skp1-F-box PPIs•Z0933M interacts with a P1 hotspot alongside C-terminal extension of Skp1•Z0933M alters SCF E3 ligase functioning, leading to substrate accumulation/modulation•Z0933M causes cell-cycle arrest, and elicits cell death by p53-dependent mechanism Drugs; Pharmaceutical science; Chemistry; Organic chemistry; Biological sciences; Cell biology S phase kinase-associated protein 1 (Skp1) is a 163-amino-acid protein that is ubiquitously expressed in different cells and tissues . StructuSkp1 depletion using genetic methods or Skp1 pharmacological inactivation reduces cancer cell viability or growth , and neoplastic transformation  of the C-terminal extension, thereby disrupting Skp1-F-box PPIs  activity and, in turn, elicits cancer cell death by p53 activation, a killing mechanism that previously has not been manifested for 6-OAP. Our findings propose Z0933M as a chemical tool compound to dissect biochemical events of Skp1 functioning in cancer biology, and a potential lead scaffold for the development of new cancer therapeutics.Taking the efforts to next stage, we herein took advantage of a hypothesis-driven, structure-guided biochemical approach to identify Z0933M as a cellularly more active Skp1 inhibitor as compared to 6-OAP. By using a series of The rationale behind the identification of Z0933M was garnered from our currently ongoing initiative to chemically exploit an aromatic cage of two phenylalanines (F101 and F139) for small-molecule drug discovery against Skp1. These phenylalanines constitute the outlet of a shallow but well-defined hotspot region, previously defined as pocket 1 (P1)  located in vitro competition assay (WT) and truncate  forms of Skp1, and by titration experiments, to characterize the specific binding of ΔSkp2F-box peptide (constructed on the consensus sequence of F-box domain of Skp2) at the F-box binding interface of Skp1 -based on assay , and S4.on assay , and theon assay C. We wer of Skp1 C. The prcreening C. At the ± 18 nM D.WT) and truncate (ΔSkp11−140) forms, in a dose-dependent fashion  values 54.7 ± 6.68 and 40.4 ± 8.2 nM, respectively. Again, 6-OAP could not show binding with ΔSkp11−140 in the SPRi evaluation , Z0933M increased the melting temperature (Tm) of recombinantly purified Skp1, both intact  protocol, we first depicted that Z0933M appears to be spatially well aligned with the P1 architecture, mainly via face-to-face (or edge-to-face) dual stacking interactions with the aromatic cage F101 and F139   plasmids to validate the results from endogenous pull-down assay  that still retained their binding capacity in our initial evaluation of P1 site-directed mutants. The resulting data revealed that although Z0933M prevented the interaction of Skp1WT with indicated F-box proteins, its ability was abolished or diminished to do so in case of Skp1F101A and Skp1F139A  , a result that is comparable to the half-maximal effective concentration (EC50) value(s) demonstrated in certain cancer cell lines including A549 cells (see below). It is also worth mentioning that increases in protein levels of certain substrates observed with the lower Z0933M concentration were generally not seen for the higher concentration (5 μM), probably owing to the higher rate of apoptosis observed at 14 and 24 h of treatment (described below).Encouraged by the above results, we next evaluated the effect of Z0933M treatment on the global levels of K48-linked polyubiquitination, a molecular feature associated with SCF E3 ligase(s)-mediated proteolysis of several cellular proteins. Side-by-side controls in this experiment included the neddylation inhibitor MLN4924 , and 6-Oclin E1) F, the inCollectively, the above-described findings suggested that Z0933M may inhibit and/or modulate the functional activity of several SCF E3 ligases and, in turn, increase the levels or alter the turnover of several substrate proteins.One expects that a potent chemical inhibitor will recapitulate the knockdown effects of its target protein, whereas overexpressing the target protein will render the cells less sensitive to the growth inhibitory effects of the compound. Thus, evaluating the effect of Z0933M treatment in context of previously reported effects of Skp1-reduced activity or by increasing the cellular level of Skp1 may constitute a simple test to confirm if Skp1 is indeed the physiologically relevant target of Z0933M.50 values: 0.58 ± 0.07 μM versus 14.09 ± 2.25 μM, respectively) in our hands (50 further dropped to double-digit nanomolar range when A549 cells were treated for 72 (EC50 = 0.099 ± 0.001 μM) and 96 h (EC50 = 0.066 ± 0.018 μM)  cell lines, hinting that probably the mechanism of cell killing by this compound solely depends upon p53 (described in next section).Knockdown experiments have previously suggested that survival of some cancer cell lines (including A549) may depend on Skp1 , whereasur hands A. Moreov.018 μM) B. EncourSkp1 knockdown and/or Skp1 pharmacological inactivation are also known to cause G2/M phase cell-cycle arrest  in both asynchronized and synchronized A549 cells  plasmids. The results showed that whereas Z0933M displayed a potent inhibitory effect on the viability of control (vector-transfected) cells in a dose-dependent manner, its killing effect was compromised or nearly completely rescued upon transfection with Flag-Skp1 WT and mutant(s) plasmids (Skp1 mutant(s), particularly Flag-Skp1F139A, compromised Z0933M ability to induce accumulation of RPA70, γ-H2AX (S139), and cleaved PARP1 , late apoptosis (Annexin V+/PI+), and early-apoptosis (Annexin V+/PI−) in a dose- and time-dependent manner (We next sought to gain insights about the cellular and molecular mechanisms for Z0933M killing of cancer cells. Z0933M treatment of A549 cells induced upregulation of γ-H2AX (S139) and cleaved PARP1 F. It, tht manner B and S8ASkp1 markedly reduced the proportions of apoptotic/necrotic cells , whereas indirect target gene (Bcl2) was downregulated. The Western blot analysis also highlighted the dose-dependent up- and down-regulation of these proteins , ATR, p-ATR (T1989), p-p53 (S15), Chk2, p-p53 (S46), Ac-p53 K370, Ac-p53 K382, and p300 I. S15 anproteins I. Collecc release  and intrinsic  apoptotic pathways. The involvement of intrinsic  apoptosis in mediating a cell death response to Z0933M was also confirmed by dose-dependent low expression of antiapopotic Bcl-2 and Bcl-xL molecules, and an increase in the level of pro-apoptotic protein BID and the cytochrome  release B. Indeed release . Subsequ release A and 6B. release C. Likewi release D. Howeve release D. These Skp1 knockdown and/or Skp1 pharmacological inhibition, such as tumor cell growth inhibition, cell-cycle arrest, and accumulation of proteins associated with replication stress. Apart from this, Z0933M may potentially induce death response attributed to pharmacological decommissioning of SCF E3 ligases  functioning and, in turn, altered the protein turnover of several substrate proteins. Moreover, we have presented different lines of evidence suggesting that Z0933M recapitulated previously reported effects of  ligases , althougin vitro biochemical and cell viability experiments  function than it did in the cellular target engagement , a few questions would be worth investigating. For instance, how Z0933M treatment alters the tethered kinetics of substrate(s) degradation ? And, whThis study characterizes the identification of a Skp1-targeting compound from a limited chemical space. Moreover, the experiments for the mechanism study have been done entirely in a human cancer cell line grown in culture, which may not reflect exactly what happens in pre-clinical animal models. In addition, we acknowledge that further work is still required to rule out the potential off-target effects involved in Z0933M functioning.liu_jinsong@gibh.ac.cn).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jinsong Liu (This work did not generate new unique reagents.IMDM) supplemented with 10% FBS (Biowest), nonessential amino acids (Gibco), penicillin–streptomycin (HyClone), and GlutaMax (Gibco). The cultured cells were maintained at 5% CO2 in humidified chamber at 37 ⁰C.HEK293, MDA-MB-231, Hs578T, MCF-7, Hela, βTC6, HepG2, LLC, HCT-116, and A-431 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum , nonessential amino acids (Gibco), penicillin–streptomycin (HyClone), and GlutaMax (Gibco). A549, H460, H1650, H1299, H358, C4-2B, Du145, LNCaP, 22RV1, Huh-7, MGC-803, MKN-7, and NCI-N87 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS (Biowest), nonessential amino acids (Gibco), penicillin–streptomycin (HyClone), and GlutaMax (Gibco). U2OS and K562 cells were cultured in Iscove’s Modified Dulbecco’s Media  in hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) or br (broad). MS data were measured on an Agilent MSD-1200 ESI-MS system.All commercially available compounds and solvents were of reagent grade and used without further treatment unless otherwise noted. Reactions were monitored by TLC using Qing Dao Hai Yang GF254 silica gel plates (5 × 10 cm); zones were detected visually under ultraviolet irradiation (254 nm) and by spraying with an ethanol solution of phosphomolybdic acid, or by fuming with iodine steam. Silica gel column chromatography was performed on silica gel (200–300 mesh) from Qing Dao Hai Yang. NMR spectra were recorded on a Bruker NMR AVANCE 400 or a Bruker NMR AVANCE 500 shown in . ChemicaS1 , 4-methoxyphenylboronic acid pinacol ester S2 , Pd(OAc)2 , PPh3 , and KOH  were dissolved in CH3CN (100 mL). The mixture was stirred at 70°C under argon atmosphere for 24 hrs. After cooled to room temperature, the solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in CH2Cl2 (200 mL) and the solution was washed with water (100 mL×3) and brine (100 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give JH-1  as a colorless solid. 1H NMR  δ 8.45 , 7.92 , 7.83 , 7.33 , 6.99 , 3.86 .4-Bromo-2-(4-methoxyphenyl)pyridine (JH-1): A mixture of 2,4-dibromopyridine JH-1 , 3-nitrophenylboronic acid S3 , Pd(PPh3)4 , and 1 M aqueous NaHCO3  were dissolved in dimethoxyethane (60 mL). The mixture was refluxed overnight under argon atmosphere. After cooled to room temperature, the solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in EtOAc (100 mL) and the solution was washed with water (50 mL×3) and brine (50 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give JH-2  as a yellow solid. 1H NMR  δ 8.70 , 8.48 , 8.25 , 7.99–7.95 , 7.82 , 7.64 , 7.35 , 6.98 , 3.83 . 13C NMR  δ 160.8, 158.1, 150.4, 148.8, 146.6, 140.4, 133.0, 131.5, 130.2, 128.3, 123.6, 122.0, 119.3, 117.6, 114.2, 55.4.3-(2-(4-Methoxyphenyl)pyridin-4-yl)aniline (JH-3): A mixture of 4-bromo-2-(4-methoxyphenyl)pyridine JH-2  in ethanol (10 mL) was added hydrazine hydrate . The reaction was stirred at 50°C for 15 min and an excess of Raney nickel slurry in water (about 1.2 equiv) was added slowly. After 1 hr, the bubbling ceased and the mixture was cooled to room temperature and filtered through Celite. The filtrate was condensed under reduced pressured, and the residue was purified by column chromatography on silica gel  to give JH-3  as a colorless solid. 1H NMR  δ 8.65 , 8.00 , 7.81 , 7.31 , 7.25 , 7.05–6.99 , 6.93 , 6.73 , 3.84 , 3.84 . 13C NMR  δ 160.5, 157.5, 149.8, 147.2, 139.8, 132.1, 130.0, 128.3, 119.6, 118.0, 117.2, 115.6, 114.1, 113.4, 55.3.To a solution of JH-3  was dissolved in Ac2O (6 mL). After stirred at room temperature under argon atmosphere for 2 hrs, the solution was condensed under reduced pressured. The crude product was then dissolved in CH2Cl2 (100 mL) and the solution was washed with saturated NaHCO3 aqueous solution (50 mL×3) and brine (50 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give JH-4  as a colorless solid. 1H NMR  δ 8.64 , 7.96 , 7.88–7.86 , 7.81 , 7.56–7.54 , 7.43–7.38 , 7.33 , 6.98 , 3.85 , 2.19 . 13C NMR  δ 168.8, 160.7, 157.9, 150.0, 148.9, 139.6, 138.9, 132.1, 129.8, 128.4, 123.0, 120.4, 119.8, 118.6, 118.2, 114.3, 55.5, 24.7. MS (ESI): m/z 319.3 [M + H]+.N-(3-(2-(4-methoxyphenyl)pyridin-4-yl)phenyl)acetamide (JH-4): 3-(2-(4-Methoxyphenyl)pyridin-4-yl)aniline JH-3 , (±)-glycidol , and Ti(OiPr)4  were dissolved in dry CH2Cl2 (10 mL). The mixture was stirred at room temperature overnight under argon atmosphere. After quenched by 10% NaOH aqueous solution, the mixture was filtered through Celite. The organic layer was dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give JH-5 ) as a colorless solid. 1H NMR  δ 8.63 , 7.96 , 7.81 , 7.34–7.27 , 7.03–6.99 , 6.88 , 6.73–6.70 , 4.02–3.98 , 3.86 , 3.81–3.77 , 3.69–3.64 , 3.37–3.33 , 3.26–3.21 . 13C NMR  δ 160.7, 157.8, 149.9, 149.8, 148.9, 140.0, 132.2, 130.2, 128.5, 119.9, 118.4, 117.0, 114.3, 113.9, 111.9, 70.5, 64.9, 55.5, 46.8. MS (ESI): m/z 349.2 [M−H]-.3-(3-(2-(4-methoxyphenyl)pyridin-4-yl)phenylamino)propane-1,2-diol (JH-5): A mixture of 3-(2-(4-Methoxyphenyl)pyridin-4-yl)aniline JH-1 , 4-acetamidophenyl boronic acid S6 , Pd(PPh3)4 , and 1 M aqueous NaHCO3  were dissolved in dimethoxyethane (10 mL). The mixture was refluxed overnight under argon atmosphere. After cooled to room temperature, the solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in EtOAc (20 mL) and the solution was washed with water (10 mL×3) and brine (10 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give JH-6  as a colorless solid. 1H NMR  δ 8.66 , 8.48 , 8.25 , 7.99–7.95 , 7.98 , 7.83 , 7.66–7.62 , 7.36 , 7.01 , 3.87 , 2.20 . 13C NMR  δ 168.6, 160.7, 157.9, 150.0, 148.6, 139.0, 134.3, 132.2, 128.4, 127.8, 120.3, 119.4, 117.8, 114.3, 55.5, 24.8. MS (ESI): m/z 317.3 [M−H]-.N-(4-(2-(4-methoxyphenyl)pyridin-4-yl)phenyl)acetamide (JH-6): A mixture of 4-bromo-2-(4-methoxyphenyl)pyridine S7 , 4-methoxyphenyl boronic acid S8 , Pd(PPh3)4 , and K2CO3  were dissolved in 9:1 (v/v) mixed dioxane and water (30 mL). The mixture was refluxed under argon atmosphere for 6 hrs. After cooled to room temperature, 4-acetamidophenyl boronic acid S6  was added. The mixture was refluxed overnight under argon atmosphere. The solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in CH2Cl2 (20 mL) and the solution was washed with water (10 mL×3) and brine (10 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give JH-7  as a colorless solid. 1H NMR  δ 7.73 , 7.60–7.44 , 7.39 , 3.86 , 2.21 . 13C NMR  δ 168.5, 159.4, 141.5, 141.1, 137.4, 133.8, 129.3, 128.4, 127.8, 125.7, 125.5, 125.4, 120.3 114.4, 55.5, 24.8. MS (ESI): m/z 340.5 [M + Na]+. benzene JH-8  was also obtained as a by-product).N-(4-(3-(4-methoxyphenyl)phenyl)phenyl)acetamide (JH-7): A mixture of 1-bromo-3-iodobenzene S1 , 3-methoxyphenylboronic acid pinacol ester S11 , Pd(OAc)2 , PPh3 , and KOH  were dissolved in CH3CN (20 mL). The mixture was stirred at 70°C under argon atmosphere for 24 hrs. After cooled to room temperature, the solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in CH2Cl2 (20 mL) and the solution was washed with water (10 mL×3) and brine (20 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give S12  as a colorless solid. 1H NMR  δ 8.47 , 7.87 , 7.56–7.54 , 7.51–7.48 , 7.38–7.34 , 6.99–6.96 , 3.87 . 13C NMR  δ 160.2, 158.7 150.3, 139.5, 133.5, 129.9, 125.4, 124.0, 119.4, 115.8, 112.3, 55.4.3-(2-(3-Methoxyphenyl)pyridin-4-yl)aniline (JH-9): A mixture of 2,4-dibromopyridine S12 , 3-nitrophenyl boronic acid S3 , Pd(PPh3)4 , and 1 M aqueous NaHCO3  were dissolved in dimethoxyethane (15 mL). The mixture was refluxed overnight under argon atmosphere. After cooled to room temperature, the solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in EtOAc (10 mL) and the solution was washed with water (10 mL×3) and brine (10 mL), dried over Na2SO4, filtered and evaporated in vacuo. The crude product S13 was dissolved in ethanol (10 mL), and hydrazine hydrate  was added. The reaction was stirred at 50°C for 15 min and an excess of Raney nickel slurry in water (about 1.2 equiv) was added slowly. After 1 hr, the bubbling ceased and the mixture was cooled to room temperature and filtered through Celite. The filtrate was condensed under reduced pressured, and the residue was purified by column chromatography on silica gel  to give JH-9  as a colorless solid. 1H NMR  δ 8.71 , 7.88 , 7.62–7.58 , 7.43–7.38 , 7.29 , 7.07 , 7.00–6.98 , 6.93 , 6.73 , 3.91 .A mixture of 4-bromo-2-(3-methoxyphenyl)pyridine S1 , 2-methoxyphenylboronic acid pinacol ester S14 , Pd(OAc)2 , PPh3 , and KOH  were dissolved in CH3CN (100 mL). The mixture was stirred at 70°C under argon atmosphere for 24 hrs. After cooled to room temperature, the solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in CH2Cl2 (50 mL) and the solution was washed with water (50 mL×3) and brine (50 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give S15  as a colorless solid. 1H NMR  δ 8.49 , 8.04 , 7.79 , 7.39–7.34 , 7.09–7.05 , 6.97 , 3.85 . 13C NMR  δ 157.3, 156.9, 149.9, 132.3, 131.2, 130.6, 128.3, 127.6, 124.8, 121.0, 111.4, 55.6.3-(2-(2-Methoxyphenyl)pyridin-4-yl)aniline (JH-10): A mixture of 2,4-dibromopyridine S15 , 3-nitrophenyl boronic acid S3 , Pd(dppf)2Cl2 , and Na2CO3  were dissolved in 1:1 (v/v) mixed dioxane and water (50 mL). The mixture was stirred at 80°C overnight under argon atmosphere. After cooled to room temperature, the solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in CH2Cl2 (50 mL) and the solution was washed with water (30 mL×3) and brine (50 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give S16  as a colorless solid. 1H NMR  δ 8.82 , 8.54 , 8.31 , 8.08 , 8.00 , 7.83 , 7.69 , 7.47–7.40 , 7.12 , 7.05 , 3.90 . MS (ESI): m/z 307.4 [M + H]+.A mixture of 4-bromo-2-(2-methoxyphenyl)pyridine S16  in ethanol (30 mL) was added hydrazine hydrate . The reaction was stirred at 50°C for 15 min and an excess of Raney nickel slurry in water (about 1.2 equiv) was added slowly. After 1 hr, the bubbling ceased and the mixture was cooled to room temperature and filtered through Celite. The filtrate was condensed under reduced pressured, and the residue was purified by column chromatography on silica gel  to give JH-10  as a yellow solid. 1H NMR  δ 8.71 , 7.98 , 7.77 , 7.40–7.37 , 7.29–7.25 , 7.11–7.02 , 6.97 , 6.76 , 3.87 , 3.80 . MS (ESI): m/z 277.4 [M + H]+.To a solution of S1 , 4-methylphenylboronic acid pinacol ester S17 , Pd(OAc)2 , PPh3 , and KOH  were dissolved in CH3CN (20 mL). The mixture was stirred at 70°C under argon atmosphere for 24 hrs. After cooled to room temperature, the solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in CH2Cl2 (20 mL) and the solution was washed with water (20 mL×3) and brine (20 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give S18  as a colorless solid. 1H NMR  δ 8.48 , 7.88–7.85 , 7.37 , 7.29–7.27 , 2.41 .N-(3-(2-(4-methylphenyl)pyridin-4-yl)phenyl)acetamide (JH-11): A mixture of 2,4-dibromopyridine S18 , 3-acetamidophenyl boronic acid S19 , Pd(PPh3)4 , and 1 M aqueous NaHCO3  were dissolved in dimethoxyethane (10 mL). The mixture was refluxed overnight under argon atmosphere. After cooled to room temperature, the solid was filtrated off and the filtrate was concentrated. The crude product was then dissolved in EtOAc (20 mL) and the solution was washed with water (10 mL×3) and brine (10 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give JH-11  as a colorless solid. 1H NMR  δ 8.66 , 8.48 , 8.25 , 7.99–7.95 , 7.98 , 7.83 , 7.66–7.62 , 7.36 , 7.01 , 3.87 , 2.20 . 13C NMR  δ 168.6, 160.7, 157.9, 150.0, 148.6, 139.0, 134.3, 132.2, 128.4, 127.8, 120.3, 119.4, 117.8, 114.3, 55.5, 24.8. MS (ESI): m/z 317.3 [M−H]-.A mixture of 4-bromo-2-(4-methylphenyl)pyridine 4Cl aqueous solution (50 mL), the organic layer was dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give S20  as a colorless solid. 1H NMR  δ 7.67 , 7.39–7.32 , 7.27–7.21 , 2.49 . MS (ESI): m/z 257.2 [M−H]-.6-phenylphenanthridine (JH-12): To a solution of 9H-fluoren-9-one  in dry THF (60 mL) was added 3 M phenyl magnesium bromide in THF . The reaction was stirred at room temperature under argon atmosphere for 1 hr. After quenched by saturated NHS20 , sodium azide , TFA , and MeSO3H  were dissolved in hexane (20 mL). The mixture was stirred at 40°C overnight under argon atmosphere. After cooled to room temperature, the reaction was quenched by 2M NaOH aqueous solution (20 mL) and filtered through Celite. The filtrate was extracted by EtOAc (30 mL) and water (20 mL×3), and the organic layer was washed by brine (10 mL), dried over Na2SO4, filtered and evaporated in vacuo. The resulting residue was purified by column chromatography on silica gel  to give JH-12  as a colorless solid. 1H NMR  δ 8.67 , 8.59 , 8.29 , 8.11 , 7.85–7.75 , 7.70–7.66 , 7.61–7.53 .A mixture of 9-phenyl-9H-fluoren-9-ol WT), truncate (ΔSkp11−140), Skp1-F-box (fused), and mutants (Skp1Mut), as a His6-fusion protein(s) in Escherichia coli BL21 (DE3) cells. All the desired plasmids were constructed using the BamH1 and HindIII restriction sites of the pETDuet-1 expression vector, and are mentioned in E.coli BL21 (DE3) and single colonies were selected on Ampicillin (100 μg mL−1) containing plates. For expressing protein, a single freshly transformed host colony was grown in a starter culture (20 mL of LB media containing 100 μg mL−1 Ampicillin) at 37°C for 15 hr. The culture was then used to inoculate 2L LB media (containing 100 μg mL−1 Ampicillin) and grown at 37°C with shaking to an OD600 of 1.0 and then cooled down to 16°C before overnight induction with 0.5 mM isopropyl-b-D-thiogalactopyranoside (IPTG). The cells were harvested by centrifugation  using a Beckman Coulter Avanti J-20 XP centrifuge.A pETDuet-1 plasmid was used for expressing different forms of Skp1, including wild type  equilibrated with buffer A. The column was washed using 20 column volumes of buffer A and the target protein (∼20 KDa) was eluted with a 5% - 40% concentration linear-gradient of buffer B . The polypeptide compositions of the fractions were monitored by 15% SDS-PAGE gel. The appropriate protein fractions were pooled and loaded onto a Source-Q column equilibrated with buffer C ) and eluted with buffer D  with a linear gradient (15% to 35%). The protein fractions were again monitored by SDS-PAGE, pooled, and gel filtered through a Superdex-75 size exclusion column equilibrated with buffer E . The peak fractions were pooled, concentrated by centrifugal ultrafiltration, and stored at −80°C. The protein concentrations were determined using the Bio-Rad dye reagent with bovine serum albumin as the standard.Cell pellets were re-suspended in lysis buffer ) (buffer A). All subsequent operations were done at 4°C. Cellular disruption was achieved by 3 to 4 passages through a Low Temperature Ultra-high Pressure Cell Disrupter JN-3000 plus  and the cell debris was removed by centrifugation at 35,000 g for 30 min. Chromatography was conducted using an Akta FPLC . The lysate was loaded onto a HisTrapMut and pETDuet-1truncate plasmids modified, from pETDuet-1WT during this study were generated by Igebio company  .The C-terminally fluorescently-labeled F-box peptide (tracer) comprising 50 amino acids (RENFPGVSWDSLPDELLLGIFSCLCLPELLKVSGVCKRWYRLASDESLWQ-FAM) was purchased from Yuanpeptide. This tracer was constructed on the consensus sequence of Skp2-F-box motif domain (PDB: 2H2PO4 prepared with two different pH (8.0 and 8.5). Data were recorded from 200 to 260 nm with a scanning speed of 20 nm/min and a bandwidth at 1.0 nm in a 0.1 cm path-length quartz cell. The resulting data was converted to per-residue molar ellipticity units, [Θ] (deg cm2 dmol−1 residue−1), and the secondary structure content was analyzed with the Dichroweb software package. The reader may refer to Since our tracer was a full F-box peptide, we first assessed its secondary structure by CD spectroscopy. CD spectra were obtained with a Jasco J-815 spectrophotometer . Samples were prepared in 2 mM NaWT or ΔSkp11−140), and FP buffer for every measurement, allowing accurate estimation of specific polarization.FP measurements were performed using an EnVision multilabel Reader (Perkin Elmer). Excitation wavelength was 485 nm, and emission was detected at 535 nm. Lyophilized F-box-FAM peptide was initially dissolved in a Tris buffer , and then a stock solution of 100 μM was prepared by subsequent dilutions in a FP buffer  glycerol, 0.001% (v/v) Triton X-100). Specific controls groups included free F-box-FAM (tracer), bound F-box-FAM in the presence of protein (Skp1D) was determined with non-linear regression and fitting to the following equation using GraphPad Prism® v6.0 . Y = Bmax ×X/(KD + X). Experiments were performed in quadruplicates in three independent experiments. In order to assure the specific binding of the tracer at the F-box binding interface of Skp1, we conducted the Skp1WT: tracer and ΔSkp11−140: tracer reactions at a fixed protein (25 μM):tracer (500 nM) ratio. The 25 μM protein concentration was selected because it gave a sufficient difference in FP values (assay window ∼76 mP) from binding saturation experiments. The other assay conditions were kept same as above. Moreover, the same experiment with the same conditions was carried out by adding previously reported Skp1 inhibitor, 6-OAP. The experiments were performed in quadruplicates in three independent experiments.The F-box-FAM peptide (0.5 μM) was incubated with the increasing amounts of Skp1 (10 nM to 100 uM) in a final volume of 20 μL binding buffer  glycerol, 0.001% (v/v) Triton X-100) in black Costar 384-well plate at room temperature for 1 hr in the dark. The fluorescence anisotropy was measured. The binding dissociation constant (Kc+ and μc−) and their standard deviations (σc+ and σc−) using the formula Z′ = 1−[3(σc+ + σc−)/Iμc+ − μc−I] (WT) and negative  controls for the FP assay, the Z′ factor is 0.73. This indicated that our Skp1:F-box-tracer FP assay could be used as a screening platform to test selected subset of ligands in this study.One important criterion reflecting the suitability of a high throughput screening (HTS) assay is the Z′ factor, which can be calculated from a number of repeated reactions to determine whether the response is large enough to obtain reliable data. In order to provide a measure of the quality of our FP assay, we calculated the Z′ factor from the mean signals of the positive and negative controls , bound Skp1WT-F-box-FAM, and FP buffer. The resultant FP values were plotted as bar graphs using ORIGIN Lab software.The selected subset of compounds were initially assayed at final concentration of 200 μM. The compounds were incubated at room temperature with Skp1WT (25 μM) and tracer (500 nM) concentrations were kept constant. The IC50 value of the compound in the gradient-concentration assay was determined with nonlinear regression and fitting using the following equation in GraphPad Prism® v6.0 . Y = Bottom + (Top – Bottom)/(1 + 10X−LogIC50). The Ki value was then calculated by using a Ki calculator  that is based on the method described previously  was kept constant at 8 μM, and the compound concentration was varied from 50 μM to 600 μM. For comparison with 6-OAP, a single concentration of 400 μM was tested with both Skp1WT and ΔSkp11−140. All reactions were set up in final volume of 10 μL in 96-well plate with 1x SYPRO Orange (Invitrogen) and incubated with compounds on ice for 30 min. According to the experimental protocol, samples were heated at 0.5°C per minute, ranging from 30 to 90°C, and the fluorescence intensity was measured at the interval of 0.5°C. Appropriate buffer blank and protein-only controls were kept, and the shift in Tm was measured as ΔTm = Tm (ligand-protein) ‒ (protein-only). The resultant ΔTm values were plotted by using GraphPad Prism® v6.0  and ORIGIN Lab softwares.TSA, also known as differential scanning fluorimetry (DSF), is used to identify the contribution of a ligand binding to an unfolding protein as a function of temperature. The detection involves increase in fluorescence of a fluorophore SYPRO Orange (Invitrogen) upon binding to the hydrophobic regions of the gradually unfolded protein. A ligand stabilizing a protein may increase the melting temperature (Tm). To detect the binding of Z0933M with Skp1, a gradient TSA was used, in which, protein samples were mixed with appropriate concentrations of ligand over the range of several folds. The protein (Skp12 (365 nm) in a UV chamber (Amersham life science). The slides were subsequently washed with DMSO, ethanol, and finally with distilled water for 15 minutes , respectively, to remove non-covalently bound compounds. After drying with nitrogen, the slides were assembled with flow cell and then mounted on SPRi instrument for measurement, or stored at 4°C to be used for later experiments.Freshly deposited gold coated standard SPRi chips/slides  were cleaned, and compound(s) at 10 mM concentration in 100% DMSO were spotted on the chips using a micropipette. After this, the slides were left for complete evaporation of DMSO (under vacuum) at room temperature for 2 hrs. After printing, the slides were exposed to UV irradiation 2.4 J/cm−1 and at 25°C. A 1:3 mixture of glycine. HCL (pH 4.2) and 0.5% (v/v) SDS, respectively, was used to regenerate the surface and remove bound proteins from the small molecules enabling the sensor chip to be reused for additional analyte injections.All the experiments were carried out using the PlexArray® HT system , which is based on surface plasmon resonance imaging. Oval regions of interests (ROIs) were set as 9 pixels × 7 pixels area in imaging area. ROIs of vehicle (DMSO) were used as controls for measurement of specific signals. All samples were injected with an association and dissociation flow rate of 3 μL sWT, ΔSkp11−140, and any of the desired mutant(s), were in the Tris buffer . For testing the compound(s), multiple concentrations  of Skp1WT or ΔSkp11−140 were flowed on the SPRi instrument as analyte to get accurate kinetic parameters. A typical sample injection cycle consists of 300 seconds association phase with the analyte solution and 400 seconds dissociation phase with running buffer at 3 μL s−1 flow rate. Other purified proteins such as mutant forms of Skp1 were tested to confirm binding pocket specificity. The highest concentration tested for each protein was 8 μM. For data analysis, we choose two software packages, ORIGIN Lab and Data Analysis Module (DAM) of Plexera. All data from SPRi reported here are after subtraction of the background intensity/signal by DAM software. In short, the entire concentration of analyte was fitted with a 1:1 Langmuir interaction integrated rate equation to obtain the kinetic constants.The Tris buffer , without any reducing agent, was used as both analyte and running buffer. Purified recombinant proteins, including Skp1Mut plasmids  form were expressed and purified by following the same protocol as mentioned above. The proteins were quantified and incubated with Z0933M overnight. For this experiment, the stock solution of Z0933M was prepared in methyl hydroxide (MeOH). After a series of procedural steps  values were calculated using GraphPad Prism® v6.0  software. The cell viability for A549 cells was also performed for 72- and 96-hrs by using the same experimental conditions as described above.For cell viability, cells were seeded in 384 opaque-walled plates with clear bottoms at 300–500 cells per well (optimum density for growth) in a total volume of 20 μL of media. The media was according to culture conditions of each individual cell line (mentioned above). After 12 hrs, compounds were added in a total volume of 5 μL of media (two-fold diluted) to each well with final concentrations from 5 nM to 30 μM. The measurements were conducted 48 hrs after seeding of the cells. Then, 25 μL of Cell-Titer GLO reagent (Promega) was added, and luminescence was measured on an EnSpire multimode plate reader (PerkinElmer), according to the manufacturer’s instructions. The estimated Cell viability with inhibitor interventions was conducted in A549 cells for 48 hrs by following the same procedure as described above, except the interventional compounds were added 1 hr before the Z0933M. The information for relevant compounds is mentioned in the WT or mutant plasmid(s) and pcDNA3.1 vector (control) for 24 hrs. Then, the media was removed, and 50 μL new media containing indicated concentrations (see For cell viability with Skp1 WT and mutant(s) overexpression, A549 cells were seeded in 96 well opaque-walled plates with clear bottoms at 35,000–40,000 cells per well in a total volume of 100 μL of media. After 12 hrs, the media was replenished and the cells were transfected with pcDNA3.1-Skp1ions see  section FRET occurs over short distances only  and, thus, represents a unique and powerful technique to monitor intermolecular protein interactions and target engagement in cells , 2017b.To develop an in-cell Skp1-F-box PPI assay, HEK293 cells were co-transfected with N-terminally mCherry-fused Skp1 (donor construct) and C-terminally copGFP-fused NIPA (acceptor construct) for 24 and 48 hrs B. To mak4 cells/well in 100 μL of DMEM containing 10% FBS were seeded in white Greiner CellStar 96-well plates (Sigma). After 12 hrs of seeding, cells were co-transfected with 0.1 μg (100 ng) of the appropriate FRET constructs  followed by incubation for 4 hrs under normal cell-growth conditions. Otherwise, the medium was removed and replaced with PBS (1 PBS washing). Plates were then analyzed on a Perkin Elmer instrument in fluorescence mode. For copGFP, excitation (Ex) was set at 482 nm and reading emission (Em) at 502 nm. For mCherry, Ex was set at 587 nm and reading Em at 610 nm. Background fluorescence was determined from wells transfected with pCDNA3.1 vector and subtracted. The FRET ratio (FR) was calculated by following equation.HEK293 cells at a density of 5 × 10c ‒ FRETb/FRETGFPc; Where FRETc and FRETGFPc are fluorescence intensities of the cells, and FRETb is the background fluorescence intensity of the wells containing media or PBS. The resulting data was analyzed in GraphPad Prism® v6.0 . The potential utility of this assay for investigating the inhibitor interventions against Skp1-F-box PPIs was affirmed by using the known Skp1 inhibitor 6-OAP as a positive control . The cells were washed using ice cold PBS, and subsequently harvested by using scrapper. The resulting suspension was centrifuged to remove the PBS and pellet the cells. The pellet was suspended in 600 μL volume of TNE lysis buffer . The resulting suspension was kept on ice for 20–30 min for complete cell lysis. Samples were homogenized using a syringe with a 0.4 mm diameter needle for 3–4 times. Resulting lysates were spun-down at 12000 xg for 10 min at 4°C. Then, the lysates were diluted with equal volume of TNE buffer and one-tenth of the supernatant was saved as protein input. For endogenous co-IP, the remaining lysate was divided into equal parts and incubated overnight at 4°C with 20 μL protein A/G Plus beads (Pierce Technology) per sample that were already pre-incubated with indicated antibodies at 4°C for 3 hrs. For flag-protein overexpressed samples, the lysates were incubated with 30 μL anti-FLAG magnetic M2 beads (Sigma-Aldrich) per sample at 4°C overnight. After immunoprecipitations, beads were washed five times with TBST  buffer to remove the non-specific bindings. Afterwards, the protein-protein complexes were eluted using elution buffer . The immunoprecipitates were then subjected to Western blotting. Detailed antibody information used for immunoblotting is mentioned in the Co-IP experiments were performed according to a reported protocol . BrieflyFor Western blotting of total protein(s), A549 cells were cultured in the absence or presence of Z0933M with various doses and at different time-points. The cells were harvested, lysed using RIPA buffer  in the presence of protease inhibitor cocktail (Roche), and quantified. The samples were loaded onto SDS-PAGE gel for separation, and transferred onto a polyvinylidene difluoride membrane (PVDF) or Nitrocellulose (NC) membrane(s) (Millipore). The membrane(s) was/were then blocked for at least 1 hr with 5% milk or Bovine Serum Albumin (BSA) in TBST ), followed by incubation with the primary antibodies at 4°C overnight. Next, the membrane(s) was/were washed three times with TBST, incubated for 2 hrs at room temperature with HRP-conjugated secondary antibodies, and washed three times again with TBST. The signal was generated with ECL , was added to accumulate polyubiquinated proteins and thus increase the sensitivity of detection. After treatment, the cells were directly lysed by the denatured buffer , a deubiquitinating enzyme inhibitor). The lysates were normalized, and then subjected to Western blotting. Detailed antibody information used for detecting K48-linked polyubiquitylated chains is mentioned in the For 5 cells/well. After 14 to 16 hrs of seeding, the cells were treated with indicated concentrations of Z0933M for 24 hrs. Then, the cells were harvested, washed twice with cold PBS, and fixed in cold 70% ethanol on ice (around 4°C) for 4 hrs. Next, the cells were washed twice with cold PBS, resuspended with cold PBS containing 20 mg/mL RnaseA (Sigma), and incubated for 30 min at 37°C. Then, PBS containing 50 mg/mL propidium iodide (PI) (Sigma) was directly added, the cells were mixed gently, and incubated for 5–10 min at room temperature in the dark. Finally, the cell-cycle phase distribution was analyzed in three different experiments using flow cytometry . At least 10,000 events/sample were acquired. Assays were conducted three times independently. The data was analyzed by FlowJo V10 software. For synchronous cell cycle analysis, cells were synchronized to G1/S boundary by a double-thymidine blockade  or indicated concentrations of Z0933M for 12 and 24 hrs. The apoptotic rates were determined by an Annexin-V/PI assay kit . After incubation for indicated time intervals, cells were harvested, washed twice with a cold PBS, resuspended in 300-500 μL of annexin-V binding buffer, and then 10 μL of annexin-V–FITC and 5 μL of PI were added. After staining in the dark for 10–15 min at room temperature, the cells were analyzed in three independent experiments using flow cytometry . The data was analyzed with FlowJo V10 software.For flow cytometric analysis of apoptotic and necrotic cells, A549 cells were seeded in six-well plates at a density of 5 × 10A549 cells were cultured in the RPMI medium supplemented with 10% FBS until they reach 80% confluency. Then the cells were treated at indicated concentrations of Z0933M for 24 hrs. Subsequently after treatment, cells were digested with 0.5% trypsin-EDTA, centrifuged, and pellet was washed once with 1 mL of PBS. Later, cells were pelleted by centrifugation for 3–5 min at 1000 rpm in 1.5 mL EP tube. After centrifugation supernatant was removed leaving the pellet that was fixed by adding 1 mL of fixation solution ). While fixation cells were continuously mixed for 30 minutes at room temperature (or store cells at 4°C for long term storage). After fixation cells were washed 4 times with 0.1M PBS (pH 7.4) for 15 min each wash. After washing, second fixation was performed by adding 0.5 mL osmium tetra-oxide (1% in PBS) and cells were incubated for 2 hrs at room temperature. After osmic fixation, cells were washed 6 times by using 0.1M PBS (pH 7.4), 15 min each wash. Dehydration of cells was performed with 30%, 50%, 70%, 80%, 90%, and 100% alcohol subsequently for 5 min each while centrifugation  at each step. After dehydration cells were washed with Acetone twice, 5 min each wash. Then cells were incubated with Acetone: resin in 3:1 for 30 min and centrifugation was performed  to remove the solution. Second round of Acetone: resin incubation was performed by using 1:1 ratio for 2–4 hrs followed by centrifugation  to remove the solution. Third round of resin incubation was performed for overnight (12 hrs) at room temperature for complete penetration of resins into the cells. After resin penetration the samples embedding was performed by osmotic polymerization for 24 hrs at 40°C. After complete polymerization the sample were cut into 1–2 μm sections. We performed the ultrathin sectioning to have a clear staining and imaging. Uranyl acetate and alkaline were used for staining the sections and subsequently imaging was performed.The activities of caspase-3/7, −8 and −9 were measured using Caspase-Glo® 3/7,8, and 9 assay kits  according to the manufacturer’s protocol. Briefly, the cells were plated over-night in a white 384 well microplate, and then treated as indicated with Z0933M. After treatment, 25 μL of each caspase substrate was added and incubated at room temperature for 30 min. Upon activation of caspase in the apoptotic cells, the substrate will give a luminescence signal after cleavage of the aminoluciferin-labelled synthetic tetrapeptide. Luminescent signals were detected using an EnSpire multimode plate reader (PerkinElmer). For inhibitor interventions and Skp1 overexpression, the experiments were performed in 96 well format.Nuclear and cytoplasmic fractionation was perfumed using Nuclear and Cytoplasmic Protein Extraction Kit (Key GEN Biotech) and according to manufacturer’s protocol.β-actin (housekeeping gene). The primers used in this study are mentioned in Total cells were extracted by using Trizol reagents as described by the manufacturer (Invitrogen). 2 μg of RNA was reverse transcribed into single stranded cDNA with a final volume of 20 μL of reaction buffer using a reverse transcription kit . The mRNA expression(s) of targeted gene(s) was/were quantified by using LightCycler® FastStart DNA Master SYBR Green I (Roche). The experiments were performed on Bio-Rad real-time PCR machine. Data were analyzed based on 2ˆΔΔCt method. Samples were run in triplicates and data were normalized based on the expression level of −1 4′,6-diamidino-2-phenylindole (DAPI) solution (Sigma) for 5 min at room temperature. Finally, the coverslips were mounted on slides with 80% glycerol and images were obtained by confocal microscope.For IF staining, the cells were cultured on 0.1% Geltrex-coated glass coverslips in six-well plates. After Z0933M treatment at indicated doses and time-interval, the cells were washed with PBS (Gibco) and fixed in 4% paraformaldehyde at room temperature for 30–60 min. The cells were then permeabilized and blocked with blocking solution , and 0.2% Triton X-100) at room temperature for 30–60 min. Primary antibodies were diluted in blocking solution and added to the cells at room temperature for 1 hr and at 4°C overnight. After three washing steps with PBS, the coverslips were incubated in appropriate secondary antibodies prepared in blocking solution for 2 hrs at room temperature. Then, cells were washed with PBS and counterstained with a 1 μg mLFor IFD, Schrodinger’s IFD protocol  was followed. Briefly, protein (Skp1) was prepared using the Protein Preparation Wizard . H-bonds were optimized, and restrained minimization was performed to relax the structure. Grid was then generated using the receptor grid generation tools. The grid box was set directly on the P1 region of Skp1, with an enclosing box of 20 Å covering the P1 residues, and other settings left as default. The compound was prepared using LigPrep . The analyses of ligand-protein interaction(s) were performed using graphical interface of Maestro  and PyMOL version 1.8.8.2.2AST) for IFD. However, in the X-ray structure of Skp1 protein, residues 37–42 and 69–84 were missing. This makes the X-ray structure of Skp1 unfit for our molecular dynamics (MD) simulations. Therefore, to solve this problem, we used the H. sapiens Skp1 sequence (Uniprot ID: P63208) and the Skp1 X-ray structure from 2AST (Chain B) as templates to generate the full-length Skp1 structure using the Prime module in . For MD simulations, the initial orientation of the Z0933M was determined from the IFD pose. The Amber ff14SB force field and the general Amber force field (GAFF) . During MD simulations, the SHAKE method was applied to constrain the covalent bonds involving the hydrogen atom (We have used the available X-ray structure of Skp1 (PDB: d (GAFF)  were used (GAFF) , which wgen atom . The Pargen atom . The cutRoot mean square deviation (RMSD) of the Cα was calculated from the MD trajectories at 100 ps intervals. Clustering is a means of partitioning data so that data points inside a cluster are more similar to each other than they are to points outside a cluster. The cluster analysis  of prote50 values are generated by non-linear regression for parameters fitting curves using GraphPad Prism.Data are expressed as mean ± SD of two or three replicates, as indicated in figure legends, and represented using GraphPad Prism. Comparisons of multiple groups were performed using 1-way ANOVA. Comparisons between 2 groups were performed using the student’s t test. All statistical calculations are included in the relevant figure legends. p values less than 0.05 were accepted as significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Dose response curve and IC


Scientific Reports 10.1038/s41598-022-20838-9, published online 06 October 2022Correction to: Cyp27a1 was incorrectly stated as Cyp27b1.The original version of this Article contained an error in Figure 1e, where the X-axis label The original Figure Additionally, the Supplementary Information Table S2 published with this Article contained an error. Under the subheading‘12α-hydroxylated BAs’, ‘Secondary BAs’,“5β-cholanic acid-3α,12α-diol-7-one ”.now reads:“5β-cholanic acid-3α,12α-diol-7-one ”.The original Article and accompanying Supplementary Information file have been corrected.