Computational analysis and in vitro evaluation of TMF 104, for its antioxidant, antimicrobial, and anticancer efficacies.

Benzylidene chromanones are small molecules, structurally similar to active phytochemicals. Herein, we report one novel benzylidene chromanone, TMF 104, for its bio-efficacies. Its computational docking for Vanin-1, antioxidant, free radical scavenging capacities, antimicrobial effects, and anticancer efficacy were analyzed. TMF 104 predicated strong binging to Vanin-1 protein with a docking energy of -8.1 kcal/mol. The compound dose-dependently exhibited free-radical scavenging and antioxidant activities when tested in vitro. The compound also had remarkable activity against Salmonella typhimurium, Enterococcus faecalis, Staphylococcus aureus, and Escherichia coli with minimum inhibitory concentration values of 1.5, 2.0, 12.5, and 13.5 µg/ml, respectively. The compound was also effective against Bacillus cereus and Pseudomonas aeruginosa albeit at higher concentrations. TMF 104 dose-dependently inhibited the proliferation of MCF-7, NCI H460, and Caki-1 cells with respective GI50 values of 24.51, 21.95, and 32.95 µg/ml, whereas the compound was toxic to normal Vero cells at much higher concentration of 264.70 µg/ml. The compound also aided in apoptosis and increased the sub G0 /G1 phase of the cell cycle in all three cancer cells tested. Our study identified a novel, potent benzylidene analogue with potent antioxidant, antimicrobial, and anticancer activities, which drives further attention for further research.

Identification of second generation benzylidene chromanone analogues as novel, potent DHODH inhibitors in acute myeloid leukemia cells.

Dihydroorotate dehydrogenase (DHODH) remains as an active target at the preclinical level against acute myeloid leukemia (AML). Herein we report potent second generation benzylidene chromanone (SBL-105) analogues to inhibit DHODH in AML cells. Virtual docking and molecular dynamic simulations were performed. Human-recombinant (rh)DHODH, THP-1, TF-1 and HL-60 cell lines were used. MTT assay was used for cell viability. Flow cytometry was used for differentiation analysis. Computational modeling and simulations predict, SBL-105 analogs bind efficiently to DHODH with improved binding energies. While all tested analogues of SBL-105 inhibited rh DHODH enzyme, SBL-105-4 and SBL-105-6 more effectively inhibited rh DHODH with an IC50 value of 3.62 and 13.61 nM respectively. SBL-105-4 exhibited excellent anti proliferative effects against THP-1, TF-1 and HL-60 cells with GI50 values of 18.78, 38.11 and 63.83 nM respectively. A similar effect was also observed in SBL-105-6 treated AML cells with respective GI50 values of 34.56, 44.40 and 38.65 nM in THP-1, TF-1 and HL-60 cells. An increase in apoptotic populations were enumerated in all three AML cells. Both these compounds also increased the differentiation marker CD11b positive populations in all the three AML cells tested. In conclusion, SBL-105-4 and SBL-105-6 were identified as potent second generation DHODH inhibitors, which drives attention for further preclinical developments.Communicated by Ramaswamy H. Sarma.

High-throughput computational screening and in vitro evaluation identifies 5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl) phenyl]-1H-isoindole-1,3(2H)-dione (C3), as a novel EGFR-HER2 dual inhibitor in gastric tumors.

Gastric cancers are caused primarily due to the activation and amplification of the EGFR or HER2 kinases resulting in cell proliferation, adhesion, angiogenesis, and metastasis. Conventional therapies are ineffective due to the intra-tumoral heterogeneity and concomitant genetic mutations. Hence, dual inhibition strategies are recommended to increase potency and reduce cytotoxicity. In this study, we have conducted computational high-throughput screening of the ChemBridge library followed by in vitro assays and identified novel selective inhibitors that have a dual impediment of EGFR/HER2 kinase activities. Diversity-based High-throughput Virtual Screening (D-HTVS) was used to screen the whole ChemBridge small molecular library against EGFR and HER2. The atomistic molecular dynamic simulation was conducted to understand the dynamics and stability of the protein-ligand complexes. EGFR/HER2 kinase enzymes, KATOIII, and Snu-5 cells were used for in vitro validations. The atomistic Molecular Dynamics simulations followed by solvent-based Gibbs binding free energy calculation of top molecules, identified compound C3 (5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl) phenyl]-1H-isoindole-1,3(2H)-dione) to have a good affinity for both EGFR and HER2. The predicted compound, C3, was promising with better binding energy, good binding pose, and optimum interactions with the EGFR and HER2 residues. C3 inhibited EGFR and HER2 kinases with IC50 values of 37.24 and 45.83 nM, respectively. The GI50 values of C3 to inhibit KATOIII and Snu-5 cells were 84.76 and 48.26 nM, respectively. Based on these findings, we conclude that the identified compound C3 showed a conceivable dual inhibitory activity on EGFR/HER2 kinase, and therefore can be considered as a plausible lead-like molecule for treating gastric cancers with minimal side effects, though testing in higher models with pharmacokinetic approach is required.

BV6 enhances apoptosis in Lung cancer cells by ameliorating caspase expressions through attenuation of XIAP, cIAP-1, and cIAP-2 proteins.

Objective: The present study aimed to investigate the inhibitory role of second mitochondria determined activator of caspases mimetic on inhibitor of apoptosis proteins (IAPs) and regulation of caspases in nonsmall cell lung cancer cell line. Materials and Methods: Dimethyl sulfoxide and 3-(4, 5-dimethyl thizol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was done to determine the IC50 of BV6 using NCI-H23 cell line. The levels of mRNA of X-linked IAP (XIAP), cellular IAP (cIAP-1), cIAP-2, caspase-6, and caspase-7 in H23 cell line were evaluated by a quantitative real-time polymerase chain reaction, while their protein expressions were tested using western blotting. Results: Two doses of BV6 dependently downregulated the expression of mRNA of XIAP (P = 0.002, P= 0.0003 vs. untreated), cIAP-1 (P = 0.05, P = 0.005 vs. untreated), and cIAP-2 (P = 0.001, P = 0.0002 vs. untreated), respectively, while the compound upregulated the mRNA expression of caspase-6 (P = 0.001, P < 0.0001 vs. untreated) and caspase-7 (P = 0.001, P = 0.0004 vs. untreated), respectively. Dose dependent of BV6 treatment significantly decreased the protein level of XIAP (P = 0.003, P = 0.007 vs. untreated), cIAP-1 (P = 0.02, P = 0.01 vs. untreated), and cIAP-2 (P = 0.008,P = 0.008 vs. untreated), respectively. However, the compound increased the protein level of caspase-6 and caspase-7 when compared to untreated control (P = 0.006,P = 0.001) and (P = 0.01, P = 0.001), respectively. Conclusions: The result showed that BV6 treatment reduced the level of mRNA of XIAP, cIAP-1, and cIAP-2 and increased the gene expression of caspase-6 and caspase-7 in NCI-H23 cell line. Therefore, the study revealed that BV6 could be used in future as additional therapeutics in lung cancer.

Insilco and Invitro approaches identify novel dual PI3K/AKT pathway inhibitors to control acute myeloid leukemia cell proliferations.

Acute myeloid leukemia (AML) is characterized by disruption of intracellular signaling due to aberration of extracellular signaling pathways, namely PI3K/AKT cascade, by dysregulating erythropoiesis and myelopoiesis. Therefore, inhibition of PI3K/AKT, either individually, or by dual inhibitors, is shown to be effective in suppression of tumorigenesis. To increase the therapeutic viability and decrease adverse effects, including cytotoxicity due to off-target kinase inhibitions, customized targeted pharmacological agents are needed that would have greater treatment potential. In this work, using an interdisciplinary approach, we have identified dual inhibitors targeted to PI3K and AKT to significantly repress the cell proliferation in AML cancers. Diversity-based high-throughput virtual screening (D-HTVS) technique followed by conventional docking approach identified small molecules from ChemBridge library, having high binding affinity for PI3KCG subunit. Further computational screening of top identified PI3K-specific lead molecules predicts dual inhibitors with high binding affinity for AKT. To rule out the possibility for cross-reaction/off-target effects of identified small molecules, lead compounds having nil or negligible binding to PI3KCA- and PI3KCB subunits were chosen. Computational screening, enzyme inhibition and cell proliferation assays show compound C16,5-{[(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)amino]methylene}-1-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione has better affinity for PI3KCG, delta, and AKT kinases compared to their respective known/established inhibitors, and has significant anti-cell proliferation activity in AML cells with a GI50 values of 77.25 nM and 49.65 nM in THP-1 and HL-60 cells, respectively. This work proposes a novel dual inhibitor that selectively targets PI3K/AKT and suppresses cell proliferation in AML cells as a potential lead molecule for treating AML cancers.

CB-RAF600E-1 exerts efficacy in vemurafenib-resistant and non-resistant-melanoma cells via dual inhibition of RAS/RAF/MEK/ERK and PI3K/Akt signaling pathways.

Background and Aim: Predicting novel dual inhibitors to combat adverse effects such as the development of resistance to vemurafenib in melanoma treatment due to the reactivation of MAPK and PI3K/AKT signaling pathways is studied to help in reversal of cancer symptoms.Reversal of cancer symptoms in melanoma associated with vemurafenib resistance is driven by reactivation of MAPK and PI3K/Akt signaling pathways. Novel dual inhibitors targeting these proteins would be beneficial to combat resistance. Methods: High-throughput virtual screening of the ChemBridge library against B-RAFV600E and Akt was performed using an automated protocol with the AutoDock VINA program. Luminescence and time-resolved fluorescence kits were used to measure enzyme activities. The MTT assay was used to determine proliferation in normal and vemurafenib-resistant A375 cells. Flow cytometry was used to examine apoptosis, cell cycle, and phosphorylation of ERK/Akt signaling pathway. Results: High-throughput screening from the ChemBridge library identified 15 compounds with high binding energy towards B-RAFV600E; among these, CB-RAF600E-1 had the highest ΔGbinding score -11.9 kcal/mol. The compound also had a high affinity towards Akt, with a ΔGbinding score of -11.5 kcal/mol. CB-RAF600E-1 dose-dependently inhibited both B-RAFV600E and Akt with IC50 values of 635 nM and 154.3 nM, respectively. The compound effectively controlled the proliferations of normal and vemurafenib-resistant A375 cells, with GI50 values of 222.3 nM and 230.5 nM, respectively. A dose-dependent increase in the sub G0/G1 phase of the cell cycle and total apoptosis was observed following compound treatment in both normal and vemurafenib-resistant melanoma cells. Treatment with CB-RAF600E-1 decreased the pERK/pAkt dual-positive populations in normal and vemurafenib-resistant A375 cells. Conclusion: CB-RAF600E-1, identified as a novel dual inhibitor effective against normal and vemurafenib-resistant melanoma cells, requires further attention for development as an effective chemotherapeutic agent for melanoma management.

GC/MS characterization and computational kinome-wide screening of pomegranate fruit extract identifies key phytochemicals interacting to CDK kinases implicated in acute myeloid leukemia cells.

Punica granatum (Pomegranate fruit) and its constituents are proven effective against various cancer types. However, a kinome-wide screening for the active phytochemicals against kinases is not reported. This study aims in validating pomegranate fruit extract (PFE) against acute myeloid leukemia (AML) cells, and computationally identifying the phytochemicals interacting with active kinases. PFE was made with Soxhlet extractor using absolute ethanol. Gas-chromatography-mass spectroscopy (GC-MS) for phytochemical identification and MTT assay for cytotoxicity in AML (THP-1, TF-1 and HL-60) cells were performed. Apoptosis, CDK5 and CDK8 were assessed with flow cytometry. Kinase profiling was performed using In silico kinome screening. GC-MS analysis revealed 38 bioactive phytochemicals in PFE including pyrazoles, aldehydes, phenols, esters, pyranosides, and octadecadienoic acids. The extract inhibited the AML cell proliferations with GI50 values of 195.5 µg/ml, 289.1 µg/ml, and 353.5 µg/ml in THP-1, THP-1, and HL-60 cells, respectively. PFE also exhibited a dose-responsive increase in apoptotic cell populations when treated to the AML cells. Computational screening and modeling predicted three critical constituents, viz., Deoxyartemisinin, 3-Methyl-3-phenyl-3H-indazole, and 8-fluoro-5,6-dimethoxy-3,4-dihydro-2H-naphthalen-1-one of pomegranate extract to interact mainly with cyclin-dependent kinases, including CDK5 and CDK8. Proteinand ligand docking predicted binding energies, and binding pose for top candidate lead molecules. In vitro assay exhibited the anticancer properties of PFE in AML cells. Computational kinome screening predicted top three PFE constituents targeting CDKs which may be responsible for the demonstrated anticancer efficacy of the extract against AML. This hypothesis further aligned with observed efficacy of PFE to inhibit CDK5 and CDK8 in all AML cells tested. PRACTICAL APPLICATIONS: Though Punica granatum (Pomegranate fruit) and its constituents are proven effective against various cancer types, a kinome-wide screening for the active phytochemicals against kinases is not reported. In this study, we have conducted GC/MS characterization of the active phytochemicals of PFE and have performed a kinome-wide screening for all the 38 identified compounds toward 310 active kinases commonly expressed in cancers. These observations warrant isolation and further evaluation of these phytochemicals or their analogues as effective CDK inhibitors against AML proliferation. Further, the computational methods used in this study will throw light on literature for new options of kinome panel screening of active phytochemicals or small molecules.

Glucose and oleic acid mediate cellular alterations in GLP-1-induced insulin-positive differentiating UCBMSCs.

Coordinated effects of glucose and oleic acid on glucagon-like peptide-1 (GLP-1) mediated differentiation of insulin-positive differentiating umbilical cord mesenchymal stromal cells (dUCBMSCs) was studied using a co-culture of NCI-H716 (GLP-1+) and UCBMSCs (insulin+). The addition of 2.5 mM glucose increased the proliferation of NCI-H716 cells by 30% and induced transformation of UCBMSCs into insulin-secreting cells in 18 days as compared to 22 days in control cells. Oleic acid (25 µM) showed decrease in cell proliferation, autophagy, and apoptosis in NCI-H716 cells while no effect was observed in dUCBMSCs. Prolonged glucose and oleic acid resulted in apoptosis and cell cycle changes in dUCBMSCs after day 18 while higher concentrations resulted in cell death. Additionally, the expression of FAS and ACC mRNA was observed in NCI-H716 and dUCBMSCs post 24-hr addition of glucose and/or oleic acid. Absorption of oleic acid was high in NCI-H716 compared to dUCBMSCs. Taken together, optimal concentrations of glucose and oleic acid could be a key factor in stimulating intrinsic GLP-1, which in turn stimulates differentiating MSCs in a glucose-dependent manner. PRACTICAL APPLICATIONS: The aim of this article was to study whether differentiating or differentiated MSCs after mobilization or post-transplant would require optimal glucose and oleic acid to naturally stimulate intrinsic GLP-1, or otherwise, the high or long-term overload of glucose or oleic acid could result in inhibition of differentiated cells resulting in failure of insulin secretion.

FNF-12, a novel benzylidene-chromanone derivative, attenuates inflammatory response in in vitro and in vivo asthma models mediated by M2-related Th2 cytokines via MAPK and NF-kB signaling.

BACKGROUND AND AIM: This study evaluates a novel benzylidene-chromanone derivative, FNF-12, for efficacy in in vitro and in vivo asthma models. METHODS: Rat basophilic leukemia (RBL-2H3) and acute monocytic leukemia (THP-1)-derived M2 macrophages were used. Human whole blood-derived neutrophils and basophils were employed. Flow cytometry was used for studying key signalling proteins. Platelet activation factor (PAF)-induced asthma model in guinea pigs was used for in vivo studies. RESULTS: The chemical structure of FNF-12 was confirmed with proton-nuclear mass resonance (NMR) and mass spectroscopy. FNF-12 controlled degranulation in RBL-2H3 cells with an IC50 value of 123.7 nM and inhibited TNF-α release from these cells in a dose-responsive way. The compound effectively controlled the migration and elastase release in activated neutrophils. IC50 value in the FcεRI-basophil activation assay was found to be 205 nM. FNF-12 controlled the release of lipopolysaccharide (LPS)-induced interleukin-10, I-309/CCL1 and MDC/CCL22 in THP-1 derived M2 macrophages. The compound suppressed LPS-induced mitogen activated protein kinase (MAPK)-p-p38 and nuclear factor kappa B(NF-kB)-p-p65 expression in these cells. A dose-dependent decrease in the accumulation of total leucocytes, eosinophils, neutrophils and macrophages was observed in PAF-induced animal models. CONCLUSION: FNF-12 was able to control the inflammatory responses in in vitro and in vivo asthma models, which may be driven by controlling M2-related Th2 cytokines via MAPK and NF-kB signaling.

A Combined Chemical, Computational, and In Vitro Approach Identifies SBL-105 as Novel DHODH Inhibitor in Acute Myeloid Leukemia Cells.

Inhibition of the dihydroorotate dehydrogenase (DHODH) has been successful at the preclinical level in controlling myeloid leukemia. However, poor clinical trials warrant the search for new potent DHODH inhibitors. Herein we present a novel DHODH inhibitor SBL-105 effective against myeloid leukemia. Chemical characteristics were identified by 1H NMR, 13C NMR, and mass spectroscopy. Virtual docking and molecular dynamic simulation analysis were performed using the automated protocol with AutoDock-VINA, GROMACS program. Human-recombinant (rh) DHODH was used for enzyme inhibition study. THP-1, TF-1, HL-60, and SKM-1 cell lines were used. MTT assay was used to assess cell viability. Flow cytometry was employed for cell cycle, apoptosis, and differentiation analysis. Chemical analysis identified the compound to be 3-benzylidene-6,7-benz-chroman-4-one (SBL-105). The compound showed high binding efficacy toward DHODH with a Gbinding score of 10.9 kcal/mol. Trajectory analysis indicated conserved interactions of SBL-105DHODH to be stable throughout the 200-ns simulation. SBL-105 inhibited rh DHODH with an IC50 value of 48.48 nM. The GI50 values of SBL-105 in controlling THP-1, TF-1, HL-60, and SKM-1 cell proliferations were 60.66, 45.33, 73.98, and 86.01 nM, respectively. A dose-dependent increase in S-phase cell cycle arrest and total apoptosis was observed by SBL-105 treatment in both cell types, which were reversed in the presence of uridine. The compound also increased the differentiation marker CD11b-positive populations in both THP-1 and TF-1 cells, which were decreased under uridine influence. SBL-105, a novel DHODH inhibitor, identified using computational and in vitro analysis, was effective in controlling AML cells and needs attention for further preclinical developments.

Structure activity evaluation and computational analysis identify potent, novel 3-benzylidene chroman-4-one analogs with anti-fungal, anti-oxidant, and anti-cancer activities.

SIGNIFICANCE: 3-Benzylidene chroman-4-ones share close homology with naturally occurring bioactive compounds. OBJECTIVES: This study evaluated the antifungal, antioxidant, and anticancer activities of novel 3-benzylidene chromanone analogs with respect to their structure-activity relationships. METHODS: Compounds 45e-64e were synthesized inhouse. Aspergillus niger (MTCC 1344) Aspergillus flavus and Botrytis cinerea were the fungal strains tested. Computational docking analysis was carried out for vanin-1, estrogen receptor (ER), and Akt proteins using Auto-dock vina. Free radical scavenging and total antioxidant capacity was analyzed using spectrophotometric methods. MCF-7 (breast cancer) cell line was used for anticancer assays. Flow cytometry was used to detect cell cycle and apoptosis. RESULTS: Out of the twenty compounds screened, compounds 47e, 50e, 52e, 57e, and 61e that possessed either methoxy and ethoxy/methyl/isopropyl group exhibited very good activity against all fungi. Compounds possessing methoxy group alone showed moderate activity and compounds devoid of methoxy, and ethoxy groups did not show any activity. When computationally analyzed against target proteins for antioxidant properties, the compounds exhibited excellent binging efficacy to vanin-1 and ERs. These predictions were translated in the in vitro free-radical scavenging and antioxidant assays. The compounds exhibited anti-proliferative efficacy in breast cancer cell line, increased the sub-G0/G1 cell cycle populations and total apoptosis in MCF-7 cells. Additionally, the compounds also depicted excelling binging energy when computationally analyzed for Akt enzyme binding. CONCLUSION: In summary, our study identified potential analogs of 3-benzylidene chroman-4-one molecules with excellent anti-fungal, anti-oxidant, and anticancer activities which demand further research for drug developments.

High-throughput virtual screening and preclinical analysis identifies CB-1, a novel potent dual B-Raf/c-Raf inhibitor, effective against wild and mutant variants of B-Raf expression in colorectal carcinoma.

Paradoxical Raf activation via Raf dimerization is a major drawback of wild/mutant B-Raf inhibitors. Herein, we report that CB-1 a novel, potent B-Raf/c-Raf dual inhibitor, effective against colon cancer cells, irrespective of their genetic status. High-throughput virtual screening of the ChemBridge library against wild B-Raf (B-RafWT), mutant B-Raf (B-RafV600E), and c-Raf was performed using an automated protocol with the AutoDock-VINA. Caco-2 and HT-29 cells were used. Of the 23,365 compounds screened computationally, CB-1 showed the highest binding energy towards B-RafWT with a ΔGbinding score of - 13.0 kcal/mol. The compound was also predicted to be effective against B-RafV600E and c-Raf molecules with ΔGbinding energies of - 10.6 and - 10.1 kcal/mol, respectively. The compound inhibited B-RafWT, B-RafV600E and c-Raf kinases with IC50 values of 27.13, 51.70, and 40.23 nM, respectively. The GI50 value of CB-1 was 247.9 nM in B-RafWT-expressing Caco-2 cells and 352.4 nM in B-RafV600E-expressing HT-29 cells. Dose-dependent increases in total apoptosis and G1 cell cycle phase arrest was observed in CB-1-treated colon cancer cells. The compound decreased B-Raf expression in both wild and mutant colon cancer cells. CB-1, a novel, potent dual B-Raf/c-Raf inhibitor was effective against colon cancer cells bearing wild-type and mutant variants of B-Raf expression.

The SMAC mimetic AT-101 exhibits anti-tumor and anti-metastasis activity in lung adenocarcinoma cells by the IAPs/ caspase-dependent apoptosis and p65-NFƙB cross-talk.

OBJECTIVES: The Inhibitors of Apoptosis (IAPs) regulate initiator and effector phases of caspase mediated apoptosis. This study evaluates the effects of SMAC mimetic AT-101 in regulation of IAPs/caspases/NFƙB-p65 in an adenocarcinoma cell line. MATERIALS AND METHODS: MTT assay was performed in the NCI-H522 cell line. Flow cytometry was used for detecting cell cycle, apoptosis, and NFƙB-p65 regulation. Effects of AT-101 on IAPs and caspases were determined by quantitative real time-PCR and western blotting. AutoDock-VINA was used for computational analysis. RESULTS: AT-101 reduced the cell proliferation of NCI-H522 with a GI50 value of 7 µM. The compound arrested adenocarcinoma cells in the G1 phase of the cell cycle and increased early and late phase apoptosis while decreasing tumor-cell trans-migration. AT-101 treatment to NCI H522 at a concentration of 0.35 µM decreased XIAP, cIAP-1, and cIAP-2 mRNA levels to 4.39±0.66, 1.93±0.26, and 2.20±0.24 folds, respectively. Increased dose of AT-101 at 0.7 µM concentration further decreased XIAP, cIAP-1, and cIAP-2 mRNA levels to 2.44±0.67, 1.46±0.93, and 0.97±0.10 folds, respectively. Similar effects of a dose-dependent decrease in the protein expressions of XIAP, cIAP-1, and cIAP-2 were observed with AT-101 treatments, while a dose-responsive increase in the mRNA and protein expression levels of caspase 6 and caspase 7 was observed in the NCI-H522 cell line. The compound exhibited binding affinity (-6.1 kcal/mol) and inhibited NFƙB-p65 in these cells. CONCLUSION: AT-101 had anti-tumor efficacy against lung adenocarcinoma cells which could be mediated through IAPs/caspase-dependent apoptosis and NFƙB-p65 cross talk. Results from this study suggests a signal cross talk between IAPs and NFkB and open new channels for further investigations in therapeutic intervention against lung cancer management.

Cassia auriculata leaf extract ameliorates diabetic nephropathy by attenuating autophagic necroptosis via RIP-1/RIP-3-p-p38MAPK signaling.

Diabetic nephropathy (DN) is the most common manifestation of high glucose induced diabetes mellitus. In this study, we report the effects of Cassia auriculata ethanol leaf extract (CALE) on DN-associated cell toxicity and complications. The effects of CALE were screened in vitro using RGE cells. Cell viability was assessed using MTT and flow cytometry. Male Sprague-Dawley rats were divided into control, DN and treatment groups (n = 8). The DN and treatment groups received 60 mg/kg/bw of streptozotocin in citrate buffer, while the treatment group was administered 150 mg/kg/bw of CALE for 10 weeks. Biochemical analysis was conducted using spectrophotometry. Kidney tissues were analyzed using hematoxylin and eosin staining and transmission electron microscopy. CD365-KIM-1 expression was assessed using flow cytometry and signalling proteins were detected using western blotting. Treatment with 30-mM glucose reduced the viability of RGE cells in a time-dependent manner and increased the population of dead RGE cells. Cotreatment with CALE reduced cell death and glucose induced protein expression of LC3-II, RIP-1 and RIP-3 in a dose-dependent manner. In addition, CALE improved the biochemical complications, renal dysfunction and pathophysiology of rats with DN and partially or fully restored the expression of key DN-associated signalling proteins, such as KIM-1 LC3-II, RIP-1, RIP-3 and p-p38MAPK in kidney cells. CALE showed protective effects, and improved DN-associated complications in RGE cells under high glucose stress conditions, potentially by inhibiting autophagic-necroptosis signals. Additionally, CALE improved the biochemical and pathological features of kidney injury while reducing autophagic-necroptosis in rat renal cells via the LC3-II-RIP-p38MAPK pathway. PRACTICAL APPLICATIONS: Results from the current investigation will add information to the literature on glucose induced renal toxicity and the protective effects of CALE over the complications of diabetic nephropathy (DN). The mechanistic investigations of the study will add light on the autophagic/necroptosis signals in DN and open new routes of investigations to study the efficacy of CALE in diabetes-related complications.

Synergistic efficacies of thymoquinone and standard antibiotics against multi-drug resistant isolates.

OBJECTIVES: To explore the antibacterial activity of thymoquinone (TQ), a quinone extracted from Nigella sativa. METHODS: This study was conducted from May 2019 to March 2020 at the Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia. The antimicrobial activity, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of TQ were determined using an agar well diffusion method and broth microdilution assays, and the synergistic effect was evaluated using antibiotics in parallel. The disruptive effect of TQ on bacterial cell membranes was determined using scanning electron microscopy. The antivirulence properties of TQ, which include adherence and biofilm formation, were also investigated using adherence and biofilm formation assays, respectively. RESULTS: Thymoquinone demonstrated bactericidal efficacy against 4/14 bacterial strains, with MIC range of 1.04-8.3 µg/mL and and MBC range of 10.41-66.66 µg/mL. Thymoquinone showed synergism against Klebsiella pneumoniae, Staphylococcus epidermidis (American Type Culture Collection 12228), Staphylococcus aureus, and Staphylococcus epidermidis in combination with the tested antibiotics. Thymoquinone inhibited bacterial adhesion by 39%-54%, 48%-68%, and 61%-81% at 0.5 × MIC, 1 × MIC, and 2 × MIC, respectively. The tested bacterial strains significantly inhibited biofilm formation after treatment with various concentrations of TQ for 24 and 48 hours. CONCLUSION: The combinatory effect of TQ with antimicrobials should be considered when developing new antimicrobial therapy regimens to overcome multidrug-resistant.

Computational and in vitro characterization of ICY-5: A potential candidate promoting mitochondrial apoptosis via the c-MET and STAT3 pathways.

Targeted chemotherapy remains the primary choice in controlling various forms of breast cancer (BC) due to its heterogenous gene expressions in various subtypes. In silico and in vitro evaluation of ICY-5, a novel arylidene analogue against c-MET, was performed. ICY-5 exhibited a docking score of -9.6 kcal/mol in inactive conformation and, - 8.6 kcal/mol in active conformation for c-MET. ICY-5 inhibited c-MET enzyme with an IC50 of 34.34 nM. The compound effectively inhibited MDA-MB 231 and MCF-7 cell proliferation, with GI50 values of 62.61 and 75.31 nM, respectively, and hepatocyte growth factor (HGF)/R c-MET phosphorylation with IC50 s of 71.41 and 83.77 nM, respectively. ICY-5 dose-dependently inhibited HGF-induced transmigration, cell scattering, invasion and altered cell cycle. An increase in apoptotic populations of these cells, with a dose-dependent decease in phosphorylation of STAT3 protein was observed. Furthermore, ICY-5 upregulated the caspase-3, caspase-9, Bcl-2-associated X and survivin, and downregulated Bcl-2, vascular endothelial growth factor, matrix metalloproteinase-2 (MMP-2), and MMP-9 in both BC cell lines. In summary, ICY-5 exhibited excellent efficacy in BC cells, targeting c-MET/SAT-3-mediated mitochondrial apoptosis. Further research will be required to ascertain ICY-5 suitability as a targeted chemotherapeutic against multiple forms of BC.

FCX-146, a potent allosteric inhibitor of Akt kinase in cancer cells: Lead optimization of the second-generation arylidene indanone scaffold.

Akt, a serine-threonine protein kinase, is regulated by class-I PI3K signaling. Akt regulates a wide variety of cell processes including cell proliferation, survival, and angiogenesis through serine/threonine phosphorylation of downstream targets including mTOR and glycogen-synthase-kinase-3-beta (GSK3ß). Targeting cancer-specific overexpression of Akt protein could be an efficient way to control cancer-cell proliferation. However, the ATP-competitive inhibitors are challenged by the highly conserved ATP binding site, and by competition with high cellular concentrations of ATP. We previously developed an allosteric inhibitor, 2-arylidene-4, 7-dimethyl indan-1-one (FXY-1) that showed promising activity against several lung cancer models. In this work, we designed a congeneric series of molecules based on FXY-1 and optimized lead based on computational, in vitro assays. Computational screening followed by enzyme-inhibition and cell-proliferation assays identified a derivative (FCX-146) as a new lead molecule with threefold greater potency than the parent compound. FCX-146 increased apoptosis in HL-60 cells, mediated in part through decreased expression of antiapoptotic Bcl-2 protein and increased levels of Bax-2 and Caspase-3. Molecular-dynamic simulations showed stable binding of FCX-146 to an allosteric (i.e., noncatalytic) pocket in Akt. Together, we propose FCX-146 as a potent second-generation arylidene indanone compound that binds to the allosteric pocket of Akt and potently inhibits its activation.

Anticancer efficacy of 3-(4-isopropyl) benzylidene-8-ethoxy, 6-methyl, chroman-4-one (SBL-060), a novel, dual, estrogen receptor-Akt kinase inhibitor in acute myeloid leukemia cells.

Estrogen receptor (ER) α is expressed in a subset of patient-derived acute myeloid leukemia (AML) cells, whereas Akt is predominantly expressed in most types of AML. Targeting AML with dual inhibitors is a novel approach to combat the disease. Herein, we examined a novel small molecule, 3-(4-isopropyl) benzylidene-8-ethoxy,6-methyl, chroman-4-one (SBL-060), capable of targeting AML cells by inhibiting ERα and Akt kinase. The chemical properties of SBL-060 were identified by proton nuclear magnetic resonance (1H-NMR), 13C-NMR, and mass spectroscopy. In silico docking was performed using an automated protocol with AutoDock-VINA. THP-1 and HL-60 cell lines were differentiated using phorbol 12-myristate 13-acetate. ERα inhibition was assessed using ELISA. The MTT assay assessed cell viability. Flow cytometry was performed for cell cycle, apoptosis, and p-Akt analyses. Chemical analysis identified the compound as 3-(4-isopropyl) benzylidene-8-ethoxy,6-methyl, chroman-4-one, which showed high binding efficacy toward ER, with a ΔGbinding score of -7.4 kcal/mol. SBL-060 inhibited ERα, exhibiting IC50 values of 448 and 374.3 nM in THP-1 and HL-60 cells, respectively. Regarding inhibited cell proliferation, GI50 values of SBL-060 were 244.1 and 189.9 nM for THP-1 and HL-60 cells, respectively. In addition, a dose-dependent increase in sub G0/G1 phase cell cycle arrest and total apoptosis was observed after treatment with SBL-060 in both cell types. SBL-060 also dose-dependently increased the p-Akt-positive populations in both THP-1 and HL-60 cells. Our results indicate that SBL-060 has excellent efficacy against differentiated AML cell types by inhibiting ER and Akt kinase, warranting further preclinical evaluations.

Computational docking and in vitro analysis identifies novel arylidene analogue FPMXY-14 against renal cancer cells by attenuating Akt.

Targeted therapies are gaining global attention to tackle Renal Cancer (RC). This study aims to screen FPMXY-14 (novel arylidene analogue) for Akt inhibition by computational and in vitro methods. FPMXY-14 was subjected to proton NMR analysis and Mass spectrum analysis. Vero, HEK-293, Caki-1, and A498 cell lines were used. Akt enzyme inhibition was studied with the fluorescent-based kit assay. Modeller 9.19, Schrodinger 2018-1, LigPrep module, and Glide docking were used in computational analysis. The nuclear status was assessed by PI/Hoechst-333258 staining, cell cycle, and apoptosis assays were performed using flow cytometry. Scratch wound and migrations assays were performed. Western blotting was applied to study key signalling proteins. FPMXY-14 selectively inhibited kidney cancer cell proliferation with GI50 values of 77.5 nM and 101.40 nM in Caki-1 cells and A-498 cells, respectively. The compound dose-dependently inhibited Akt enzyme with an IC50 value of 148.5 nM and bound efficiently at the allosteric pocking of the Akt when computationally analyzed. FPMXY-14 caused nuclear condensation/fragmentation, increased the sub G0/G1, G2M populations, and induced early, late phase apoptosis in both cells when compared to controls. Treatment of the compound inhibited wound healing and migration of tumor cells, while proteins like Bcl-2, Bax, and caspase 3 were also altered. FPMXY-14 effectively inhibited the phosphorylation of Akt in these cancer cells, while total Akt was unaltered. FPMXY-14 exhibited anti-proliferative and anti-metastatic activities in kidney cancer cells by attenuating the Akt enzyme. Further pre-clinical research on animals with a detailed pathway elucidation is recommended.

Computational high-throughput screening and in vitro approaches identify CB-006-3; A novel PI3K-BRAFV600E dual targeted inhibitor against melanoma.

Malignant melanoma is characterized by both genetic and molecular alterations that activate phosphoinositide 3-kinase (PI3K), and RAS/BRAF pathways. In this work, through diversity-based high-throughput virtual screening we identified a lead molecule that selectively targets PI3K and BRAFV600E kinases. Computational screening, Molecular dynamics simulation and MMPBSA calculations were performed. PI3K and BRAFV600E kinase inhibition was done. A375 and G-361 cells were used for in vitro cellular analysis to determine antiproliferative effects, annexin V binding, nuclear fragmentation and cell cycle analysis. Computational screening of small molecules indicates compound CB-006-3 selectively targets PI3KCG (gamma subunit), PI3KCD (delta subunit) and BRAFV600E. Molecular dynamics simulation and MMPBSA bases binding free energy calculations predict a stable binding of CB-006-3 to the active sites of PI3K and BRAFV600E. The compound effectively inhibited PI3KCG, PI3KCD and BRAFV600E kinases with respective IC50 values of 75.80, 160.10 and 70.84 nM. CB-006-3 controlled the proliferation of A375 and G-361 cells with GI50 values of 223.3 and 143.6 nM, respectively. A dose dependent increase in apoptotic cell population and sub G0/G1 phase of cell cycle were also observed with the compound treatment in addition to observed nuclear fragmentation in these cells. Furthermore, CB-006-3 inhibited BRAFV600E, PI3KCD and PI3KCG in both melanoma cells. Collectively, based on the computational modeling and in vitro validations, we propose CB-006-3 as a lead candidate for selectively targeting PI3K and mutant BRAFV600E to inhibit melanoma cell proliferation. Further experimental validations, including pharmacokinetic evaluations in mouse models will identify the druggability of the proposed lead candidate for further development as a therapeutic agent for treating melanoma.