Multiple Ligand Simultaneous Docking Research Articles

Pharmacokinetic predictions of ROS-mediated targets and genotoxin combinations via multiple ligand simultaneous docking and ROS evaluation in vitro using HepG2 cell lines.

Although combination therapy is known for its high efficacy, reduced side effects and drug resistance, toxicity remains a major drawback. Some of the genes are likely to induce hepatotoxicity through ROS-mediated mechanisms when a drug is metabolized alone or in combination in the liver. To address this, we have developed a scientific approach to predict the toxicity of different genotoxin combinations and validate their interactions with various targets. The current study is an extensive study of our previous set of in vivo rat liver microarray data processed using R studio for their functional analysis. About five combinations of genotoxins such as CPT/ETP, CPT/CPL, ETP/CPL, CP/CPT and EES/CP along with their differential gene expression targeting Chemical carcinogenesis-ROS are chosen for this study. We aim to examine the binding affinity of different genotoxin combinations using in silico multiple ligand simultaneous docking (MLSD) and are then bio-evaluated for cytotoxicity in vitro using human hepatocellular carcinoma cell lines (HepG2) with the MTT assay. As a result, dose-response cytotoxicity with its strength of interactions and a significant variance in ROS levels in the treated cells is observed compared to their IC50 values. Out of 5 combinations such as CPT/CPL, ETP/CPL and EES/CP are found not only to be significantly cytotoxic but also induce oxidative stress specifically above their IC50 values with good and moderate binding interactions ensuring their toxicity. On the contrary, the safe combinations are found to be CTP/ETP and CP/CPT possibly with no and tolerable adverse effects standing as preliminary information for researchers in drug design and development.

The interactions of antiviral drugs and a phosphonium fluorescent dye with proteins as revealed by a multiple ligand simultaneous docking

In the present study, the multiple ligands simultaneous docking technique was employed to explore the feasibility of developing the protein-based nanocarriers in which the therapeutic agents (antiviral drugs) will be combined with a visualizing agent (the phosphonium dye TDV). By varying both the protein (serum albumin, lyso-zyme, cytochrome c, insulin) and antiviral agent (favipiravir, molnupiravir, nirmatrelvir, ritonavir) components of the examined complexes it was demonstrated that in the albumin- and cytochrome c-containing systems the TDV and antiviral dyes occupy distinct binding sites on the protein molecule, exhibiting no interference with each other. It was found that the TDV propensity to reside within the same insulin cavity as the antiviral agent might hinder the drug’s release from the nanocarrier. Our results indicate that the most promising energetically stable protein-drug-TDV systems for developing protein-based nanocarriers with the examined antiviral drugs can be created on a basis of serum albumin.

IN SILICO STUDY OF THE SYNERGISTIC INTERACTION OF 5-FLUOROURACIL AND CURCUMIN ANALOGUES AS INHIBITORS OF B-CELL LYMPHOMA 2 PROTEIN

Objective: The research objective is to explore the potential for synergistic inhibition of two curcumin analogs combined with 5-fluorouracil (5-FU) against the B-cell lymphoma 2 (BCL-2) protein.
 Methods: We investigated the synergistic inhibition of two curcumin analogs, namely, (1E,4E)-1,5-bis(4-hydroxyphenyl)penta-1,4-dien-3-one (AC01) and (1E,4E)-1,5-bis(3,4-dihydroxyphenyl)penta-1,4-dien-3-one (AC02), each combined with 5-FU by calculating their binding free energies and binding stability. An in silico investigation of the synergistic interaction of ligand pairs was conducted using the multiple ligand simultaneous docking (MLSD) technique with the AutoDock Vina package. The stability of interactions and binding free energies of each BCL-2 and curcumin analogs were examined by applying molecular dynamics techniques with the Gromacs package and MMPBSA method.
 Results: All ligand pairs had displayed strong binding affinity, as evidenced by highly negative free energy values, indicating a robust association with BCL-2. Molecular dynamics simulations were conducted over 100 ns, confirming good stability with controlled RMSD changes, suggesting that the ligand pairs had remained securely bound to the BCL-2 binding site. Additionally, RMSF analysis and energy decomposition had revealed that ligand interactions did not influence protein residue fluctuations during the simulation, and the protein-ligand complexes had maintained stability throughout the simulation. Furthermore, binding free energy calculations using the MMPBSA method had consistently shown negative values, signifying stable interactions with BCL-2 for all ligand pairs.
 Conclusion: In conclusion, our study revealed that AC01 and AC02, when combined with 5-FU, had the ability to intercalate into the P2 and P4 sites of BCL-2. This suggested that AC01 and AC02 held promise for further study as candidates for anticancer drugs, individually or in combination with 5-FU.

“From shadows to shores”-quantitative analysis of CuO nanoparticle-induced apoptosis and DNA damage in fish erythrocytes: A multimodal approach combining experimental, image-based quantification, docking and molecular dynamics

The usage of copper (II) oxide nanoparticles (CuO NPs) has significantly expanded across industries and biomedical fields. However, the potential toxic effects on non-target organisms and humans lack comprehensive understanding due to limited research on molecular mechanisms. With this study, by combining the 96 h in vivo exposure of crucian carp fish, Carassius carassius, to sub-lethal CuO NPs doses (0.5 and 1 mg/dL) with image-based quantification, and docking and molecular dynamics approaches, we aimed to understand the mechanism of CuO NPs-induced cyto-genotoxicity in the fish erythrocytes. The results revealed that both doses of copper NPs used were toxic to erythrocytes causing oxidative stress response and serious red blood cell morphological abnormalities, and genotoxicity. Docking and 10-ns molecular dynamics confirmed favorable interactions (ΔG = −2.07 kcal mol−1) and structural stability of Band3-CuO NP complex, mainly through formation of H-bonds, implying the potential of CuO NPs to induce mitotic nuclear abnormalities in C. carassius erythrocytes via Band3 inhibition. Moreover, conventional and multiple ligand simultaneous docking with DNA revealed that single, double and triple CuO NPs bind preferentially to AT-rich regions consistently in the minor grooves of DNA. Of note, the DNA-binding strength subtantially increased (ΔG = −2.13 kcal mol−1, ΔG = −4.08 kcal mol−1, and ΔG = −6.03 kcal mol−1, respectively) with an increasing number of docked CuO NPs, suggesting that direct structural perturbation on DNA could also count for the molecular basis of in-vivo induced DNA damage in C. carassius erythrocytes. This study introduces the novel term “erythrotope” to describe comprehensive red blood cell morphological abnormalities. It proves to be a reliable and cost-effective biomarker for evaluating allostatic erythrocyte load in response to metallic nanoparticle exposure, serving as a distinctive fingerprint to assess fish erythrocyte health and physiological fitness.

Multiple Ligand Simultaneous Docking of Antiviral Drugs and Cyanine Dyes with Proteins

Protein nanoparticles are currently regarded as promising biocompatible and biodegradable systems for targeted delivery of different types of pharmacological agents. Prior to fabricating such kind of drug nanocarriers it is reasonable to evaluate the drug-protein binding affinity and possible interaction modes using the computational tools, particularly, the molecular docking technique. The present study was undertaken to evaluate the possibility of creating the protein nanoparticles carrying the antiviral drugs and cyanine dyes as visualizing agents. The components of the examined systems included endogenous functional proteins cytochrome c, serum albumin, lysozyme and insulin, antiviral drugs favipiravir, molnupiravir, nirmatrelvir and ritonavir, mono- and heptamethinecyanine dyes. Using the multiple ligand simultaneous docking technique, it was demonstrated that: i) the drugs and the dyes occupy different binding sites on the protein molecule and do not interfere with each other; ii) the heptamethines AK7-5 and AK7-6 possess the highest affinity for the proteins; iii) among the examined systems the strongest complexes are formed between the heptamethine dyes and serum albumin. Taken together, the results obtained indicate that albumin-based nanoparticles functionalized by the heptamethine cyanine dyes can be used for targeted delivery of the explored antiviral agents.

Biocomputational-mediated screening and molecular docking platforms for discovery of coumarin-derived antimelanogenesis agents

Background: Hyperpigmentation occurs when excess melanin accumulates in the skin and causes the skin to become darker in color. Pursuing attractive appearance and colorism have promoted the development of the skin whitening market globally. The proteins targeted in this research are tyrosinase-related protein 1, cyclic adenosine monophosphate response element-binding protein, receptor tyrosine kinase, and endothelin receptor type B. Objectives: This study aims to identify the potential of coumarin derivatives as novel effective, safe, and natural antimelanogenesis agents for whitening purposes or therapeutical intention to treat hyperpigmentation disorders. Methods: Four three-dimensional structures of the targeted proteins and 94 ligands were obtained from Protein Data Bank and PubChem, respectively. The ligands were docked against modified targeted proteins to examine the binding affinity and protein-ligand interactions using PyRx and BIOVIA Discovery Studio. The top 13 derivatives were selected for further analysis on the pharmacokinetic properties through SwissADME and pkCSM web servers. A total of eight compounds were further chosen to conduct multiple ligand simultaneous docking (MLSD). Results: Difenacoum is the most potential antimelanogenesis agent due to its strong inhibitory binding affinity in targeted protein models (5M8M, 4TQN, 5X93), but it does not exhibit favorable behavior pharmacokinetic properties. From the in silico pharmacokinetics screening, novobiocin sodium is the most potent derivative due to its relatively appropriate and safer properties. However, none of the ligand pairs investigated in MLSD possesses a synergistic effect on the binding affinity. Conclusion: Our findings identified colladin, farnesiferol C and novobiocin sodium may be promising natural resources for developing antimelanogenesis agents.

Comparative computational study to augment UbiA prenyltransferases inherent in purple photosynthetic bacteria cultured from mangrove microbial mats in Qatar for coenzyme Q10 biosynthesis.

Coenzyme Q10 (CoQ10) is a powerful antioxidant with a myriad of applications in healthcare and cosmetic industries. The most effective route of CoQ10 production is microbial biosynthesis. In this study, four CoQ10 biosynthesizing purple photosynthetic bacteria: Rhodobacter blasticus, Rhodovulum adriaticum, Afifella pfennigii and Rhodovulum marinum, were identified using 16S rRNA sequencing of enriched microbial mat samples obtained from Purple Island mangroves (Qatar). The membrane bound enzyme 4-hydroxybenzoate octaprenyltransferase (UbiA) is pivotal for bacterial biosynthesis of CoQ10. The identified bacteria could be inducted as efficient industrial bio-synthesizers of CoQ10 by engineering their UbiA enzymes. Therefore, the mutation sites and substitution residues for potential functional enhancement were determined by comparative computational study. Two mutation sites were identified within the two conserved Asp-rich motifs, and the effect of proposed mutations in substrate binding affinity of the UbiA enzymes was assessed using multiple ligand simultaneous docking (MLSD) studies, as a groundwork for experimental studies.

Computational screening for investigating the synergistic regulatory potential of drugs and phytochemicals in combination with 2-deoxy-D-glucose against SARS-CoV-2.

COVID-19 disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was declared a global pandemic by the World Health Organization (WHO) in March 2020. Since then, the SARS-CoV-2 virus has impacted millions of lives worldwide. Various preclinical and clinical trials on the treatment of COVID-19 disease have revealed that the drugs that work in combination are more likely to reduce reinfection and multi-organ failure. Considering the combination drug therapy, herein, we performed a systematic computational study starting with the formation of sixty-two combinations of drugs and phytochemicals with 2-deoxy-D-glucose (2-DG). The top nineteen combinations resulting from Drug-Drug Interaction (DDI) analysis were selected for individual and multiple-ligand-simultaneous docking (MLSD) study with a host target Serine Protease (TMPRSS2; PDB ID: 7MEQ) and two viral targets, Main Protease (3CLpro; PDB ID: 6LU7) and Uridylate-Specific Endoribonuclease (NSP15; PDB ID: 6VWW). We found that the resulting drugs and phytochemicals in combination with 2-DG shows better binding than the individual compounds. We performed the re-docking of the top three drug combinations by utilizing the polypharmacology approach to validate the binding patterns of drug combinations with multiple targets for verifying the best drug combinatorial output obtained by blind docking. A strong binding affinity pattern was observed for 2-DG + Ruxolitinib (NIH-recommended drug), 2-DG + Telmisartan (phase 4 clinical trial drug), and 2-DG + Punicalagin (phytochemical) for all the selected targets. Additionally, we conducted multiple-ligand-simultaneous molecular dynamics (MLS-MD) simulations on the selected targets with the 2-DG + Ruxolitinib combination. The MLS-MD analysis of the drug combinations shows that stabilization of the interaction complexes could have significant inhibition potential against SARS CoV-2. This study provides an insight into developing drug combinations utilizing integrated computational approaches to uncover their potential in synergistic drug therapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11224-022-02049-0.

Effect of chlorogenic acid plus donepezil on critical neurocortical enzyme activities, inflammatory markers, and synaptophysin immunoreactivity in scopolamine-assaulted rats, supported by multiple ligand simultaneous docking.

The effect of chlorogenic acid (a natural phenolic acid ubiquitous in plant foods) on selected therapeutic properties of donepezil (DON) in a scopolamine (SCOP)-induced rat model of amnesia was the focus of this study. Adult albino (Wister strain) rats were allocated into five groups (n=11) consisting of control, SCOP, SCOP + chlorogenic acid (CGA), SCOP + DON, and SCOP + CGA + DON for 7 days. Post-treatment, the rat brain cerebral cortex homogenate was assayed for cholinesterase and monoamine oxidase activities. Also, the reactive oxygen species, total thiol and nitric oxide contents, alongside catalase, and superoxide dismutase activities were determined. Routine histology for neuronal and glial cells as well as synaptophysin immunoreactivity was also carried out on the cerebral cortex. Thereafter, multiple ligand simultaneous docking was carried out for DON and CGA at the active sites of AChE and BChE. The results revealed that the biochemical parameters, glial cells, and synaptophysin immunoreactivity were significantly impaired in the cerebral cortex of scopolamine-treated rats. However, impaired butyrylcholinesterase and monoamine oxidase activity, together with antioxidant, glial cells, and synaptophysin levels were significantly ameliorated in scopolamine-treated rats administered DON + CGA compared to donepezil alone. The docking of both DON and CGA at the active sites of AChE or BChE showed higher binding energy to both enzymes compared to individual interactions of either DON or CGA. Hence, this study has been able to show that CGA could improve some of the therapeutic effects of DON, which could broaden the therapeutic spectrum of this drug. PRACTICAL APPLICATIONS: This study showed that chlorogenic acid (a major phenolic acid found in plant foods such as coffee) modulated some of the therapeutic properties of donepezil (an anticholinesterase drug used in the treatment of mild-to-moderate Alzheimer's disease). The combinations elicited better anti-butyrylcholinesterase, antimonoamine oxidase, and antioxidant properties, thus presenting this food-drug interaction as potentially able to offer better therapeutic properties.

Bazedoxifene, a GP130 Inhibitor, Modulates EMT Signaling and Exhibits Antitumor Effects in HPV-Positive Cervical Cancer.

Persistent HPV (Human Papillomavirus) infection is the primary cause of cervical cancer. Despite the development of the HPV vaccine to prevent infections, cervical cancer is still a fatal malignant tumor and metastatic disease, and it is often difficult to treat, so a new treatment strategy is needed. The FDA-approved drug Bazedoxifene is a novel inhibitor of protein–protein interactions between IL-6 and GP130. Multiple ligand simultaneous docking and drug repositioning approaches have demonstrated that an IL-6/GP130 inhibitor can act as a selective estrogen modulator. However, the molecular basis for GP130 activation in cervical cancer remains unclear. In this study, we investigated the anticancer properties of Bazedoxifene in HPV-positive cervical cancer cells. In vitro and in vivo experiments showed that Bazedoxifene inhibited cell invasion, migration, colony formation, and tumor growth in cervical cancer cells. We also confirmed that Bazedoxifene inhibits the GP130/STAT3 pathway and suppresses the EMT (Epithelial-mesenchymal transition) sub-signal. Thus, these data not only suggest a molecular mechanism by which the GP130/STAT3 pathway may promote cancer, but also may provide a basis for cervical cancer replacement therapy.

A novel small molecule LLL12B inhibits STAT3 signaling and sensitizes ovarian cancer cell to paclitaxel and cisplatin.

Ovarian cancer is the fifth most common cause of cancer deaths among American women. Platinum and taxane combination chemotherapy represents the first-line approach for ovarian cancer, but treatment success is often limited by chemoresistance. Therefore, it is necessary to find new drugs to sensitize ovarian cancer cells to chemotherapy. Persistent activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling plays an important role in oncogenesis. Using a novel approach called advanced multiple ligand simultaneous docking (AMLSD), we developed a novel nonpeptide small molecule, LLL12B, which targets the STAT3 pathway. In this study, LLL12B inhibited STAT3 phosphorylation (tyrosine 705) and the expression of its downstream targets, which are associated with cancer cell proliferation and survival. We showed that LLL12B also inhibits cell viability, migration, and proliferation in human ovarian cancer cells. LLL12B combined with either paclitaxel or with cisplatin demonstrated synergistic inhibitory effects relative to monotherapy in inhibiting cell viability and LLL12B-paclitaxel or LLL12B-cisplatin combination exhibited greater inhibitory effects than cisplatin-paclitaxel combination in ovarian cancer cells. Furthermore, LLL12B-paclitaxel or LLL12B-cisplatin combination showed more significant in inhibiting cell migration and growth than monotherapy in ovarian cancer cells. In summary, our results support the novel small molecule LLL12B as a potent STAT3 inhibitor in human ovarian cancer cells and suggest that LLL12B in combination with the current front-line chemotherapeutic drugs cisplatin and paclitaxel may represent a promising approach for ovarian cancer therapy.

488-P: Isoliquiritigenin Protects Renal Tubular Epithelial Cells from Inflammatory and Oxidative Injuries via Directly Activating SIRT1 in Type 1 Diabetic Nephropathy

Emerging evidence shows that increasing fibrosis and apoptosis have been observed in the tubular epithelia in the progression of type 1 diabetic nephropathy (DN). Excessive inflammatory response and oxidative stress contribute to the pathogenesis of progression. Isoliquiritigenin (ISL), a natural chalcone compound, has been reported to exert anti-inflammatory and anti-oxidative properties. However, little is known about the effects of ISL on diabetic nephropathy and its mechanism. In this study, we investigated the potential protective effects and pharmacological mechanisms of ISL on DN in vivo and in vitro. We test beneficial activities of ISL on streptozotocin (STZ)-induced type 1 diabetes mellitus mice and renal tubular epithelial NRK-52E cells. The study showed that ISL attenuated diabetic nephropathy by decreasing both pro-fibrotic and pro-apoptotic biomarkers. Diabetes or high glucose (HG) induced inflammatory response and oxidative stress were eliminated by ISL administration. The molecular docking using the multiple ligand simultaneous docking (MLSD) software indicated that three molecules of ISL might bound to the binding site of SIRT1 and formation of a stable complex. We also found ISL could neutralize HG-mediated inactivation of SIRT1. Activation of SIRT1 could attenuate HG-induced inflammation, oxidative stress, fibrosis and apoptosis in NRK-52E cells, paralleled with the effects of ISL. While inhibition of SIRT1 could significantly abrogate the protective effects of ISL. Our study implicated that ISL might have great pharmacological actions on attenuating tubular damage of diabetic nephropathy. The beneficial effects of ISL are likely associated with inhibiting excessive inflammatory response and oxidative stress via directly activating SIRT1. Disclosure X. Chen: None. F. Shen: None. Funding Wenzhou Science and Technology Bureau (Y20190125); First Affiliated Hospital of Wenzhou Medical University (FHY2019015)

In silico and in vitro analysis reveal multi-target anti-hyperglycaemic properties of gedunin, a limonoid from neem (Azadirachta indica)

BackgroundInsulin secretion and insulin related pathways have been the prime targets in the treatment of diabetes for a long time. However, recently a lot of attention is being directed towards addressing hyperglycaemia as the main perpetrator of the symptoms in this metabolic disorder. This new treatment approach also involves greater inclination to plant derived therapeutic agents for their safety and probable minimal side effects. The objective of the present study was to scientifically elucidate the potential of gedunin (a limonoid from Neem tree) as an anti-hyperglycaemic agent.MethodsThe effect of gedunin on pancreatic and salivary amylase activity and glucose transport across yeast cell membrane was tested at three different concentrations (5 μM, 10 μM and 20 μM) using known inhibitor acarbose as the standard. Multiple Ligand Simultaneous Docking was used to study the interaction of gedunin with salivary and pancreatic amylase and determine binding affinity and specificity of this interaction.ResultsThe in vitro results documented a steady, linear pancreatic alpha amylase (ovine) inhibition in a concentration dependent manner with gedunin showing lower IC50 value of 20.25 μM against acarbose (IC50 = 31.12 μM) a known enzyme inhibitor used as standard in the present study. The inhibition of salivary amylase by gedunin was also distinct. Yeast cell glucose uptake studies revealed remarkable inhibition of glucose absorption at 10 μM and 20 μM concentration of gedunin (5.45% and 13.87% respectively with respect to control). Corroborating the in vitro findings even in the docking studies gedunin exhibited higher docking score (− 8.12 Kcal/mol) and higher enzyme inhibition potency (Ki = 1.12 μM) with human pancreatic amylase-substrate complex as compared to acarbose (docking score-5.24 Kcal/mol, Ki = 110.8 μM). The studies further suggested a non-competitive, mixed kind of inhibition by gedunin. As evident from this current in vitro study, gedunin had shown significant inhibition of alpha amylases and glucose uptake at much lower concentration (5, 10 and 20 μM) than previous studies where the concentrations used were (20.7–124.3 μM).ConclusionThis study lays strong evidence to the rationale of gedunin being an important lead compound to developing a promising hyperglycaemic agent, simultaneously targeting glucose absorption in the intestine and enzymatic digestion of polysaccharides.

LLY17, a novel small molecule STAT3 inhibitor induces apoptosis and suppresses cell migration and tumor growth in triple-negative breast cancer.

Persistent STAT3 signaling is frequently detected in many cancer types including triple-negative breast cancer, and thus could potentially serve as a viable therapeutic target. We have designed a novel non-peptide compound LLY17 targeting STAT3 using Advanced Multiple Ligand Simultaneous Docking (AMLSD) methods. However, the efficacy of LLY17 has not been evaluated extensively in human and murine triple-negative breast cancer cells. In this study, we tested LLY17 in multiple human and murine triple-negative breast cancer cell lines. Human triple-negative breast cancer MDA-MB-468, MDA-MB-231, SUM159, and BT-549 cells, and murine triple-negative breast cancer 4T1 cells were used to study the inhibition effects of LLY17. The inhibition of STAT3 activation of LLY17 was investigated using western blot analysis. Cell viability, apoptosis and migration assays were carried out by MTT assay, Caspase-3/7 assay and wound healing assay, respectively. A mammary fat pad syngeneic mouse model was used to evaluate the antitumor effect of LLY17 in vivo. LLY17 inhibited IL-6-mediated induction of STAT3 phosphorylation but had no effect on IFN-γ-induced STAT1 phosphorylation or EGF-induced ERK phosphorylation. LLY17 inhibited STAT3 phosphorylation and induced apoptosis in human and murine triple-negative breast cancer cells but exhibited minimal toxicity toward Luminal A subtype breast cancer MCF-7 cells. RNAi attenuation experiments supported the requirement of STAT3 for LLY17-mediated inhibition of cell viability in triple-negative breast cancer cells. In addition, LLY17 inhibited cell migration of human and murine triple-negative breast cancer cells. Furthermore, LLY17 suppressed tumor growth and STAT3 phosphorylation of triple-negative breast cancer cells in a mammary fat pad syngeneic mouse model in vivo. Together, our findings suggest that targeting persistent STAT3 signaling by novel small molecule LLY17 may be a potential approach for the therapy of triple-negative breast cancer.

Curcumin and capsaicin modulates LPS induced expression of COX-2, IL-6 and TGF-β in human peripheral blood mononuclear cells.

The mechanism of action of treatment of either curcumin or capsaicin or in combination on LPS (Lipopolysaccharide) induced inflammatory gene expression in peripheral blood mononuclear cells (PBMCs) was investigated using RT-PCR and in silico docking methods. RT-PCR analysis has shown that the curcumin and capsaicin significantly reduced LPS induced over expression of COX-2, IL-6 and TGF-β in PBMCs. Whereas combined molecules demonstrated synergistic response on the reduction of COX-2, IL-6 and TGF-β over expression in LPS induced PBMCs as compared to individual molecules. Further, The docking of curcumin and capsaicin at the active pockets of COX-2, IL-6 and TGF-β has shown - 3.90, - 4.49 and - 5.61kcal/mol binding energy for curcumin and - 3.80, - 4.78 and - 5.76kcal/mol binding energy for capsaicin, while multiple ligand simultaneous docking (MLSD) of both molecules has shown higher binding energy of - 4.24, - 5.35 and - 5.83kcal/mol respectively. This has demonstrated the efficacy of combined curcumin and capsaicin against the LPS induced expression of pro-inflammatory cytokines in PBMCs. These results attributed the coordinated positive modulation on biochemical and molecular cellular process by combined curcumin and capsaicin as compared to individual molecules.

Understanding the binding interaction in therapeutic prevention of inflammation by controlling Hsp90 through curcumin and epigallocatechin

Inflammatory checkpoint blockade is the trend of current research targeting Hsp90 with either phytochemical or commercial drugs. It has been extensively studied globally by researchers but nevertheless has been unable to prevent inflammatory disorders successfully. These key considerations set the stage for identification of different combinatorial strategies, curcumin and epigallocatechin to target Hsp90 involved in inflammatory disorders to gain insight into the benefits of this approach. The primary objective in present study is the prior prediction of the binding interactions of curcumin and epigallocatechin with Hsp90 in the living system through computational studies. A suitable animal model sequence for Hsp90 studies was fetched through PHYLOGENY and BLAST. Hsp90 Mus musculus was obtained as a homologous sequence to Hsp90 Homo sapiens and its tertiary structure was homology modeled as its unabridged experimentally derived structure was not available. In additional tertiary model structure was verified using different tools. Single ligand docking and multiple ligand simultaneous docking of curcumin and epigallocatechin to the modeled Hsp90 structure was carried out using the AutoDock tool. In single ligand docking, curcumin had the binding affinity of − 8.3 kcal/mol, while for epigallocatechin − 5.9 kcal/mol and the multiple ligand simultaneous docking binding affinity for Hsp90 to curcumin and epigallocatechin was − 8.4 kcal/mol. Their interface residues were explored for mutational studies and were found to be in an unstabilized state, possibly corresponding to the conformational changes in Hsp90. These strategies emphasize the rationale for combining the modalities, curcumin and epigallocatechin in the successful reduction of Hsp90 expression, henceforth the inflammatory checkpoint would be controlled to augment the anti-inflammatory response.

Abstract 864: Repurposing FDA-approved drug bazedoxifene as a novel inhibitor of IL-6 signaling for triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is a major cause of death among breast cancer patients and is the only subtype of breast cancer that is still lacking effective therapeutic options. IL-6 is one of the principal oncogenic mediators in breast cancer and systemic IL-6 levels correlates with poor prognosis, advanced disease, and metastases. Importantly, growth of TNBC cells are replied on autocrine cytokines including IL-6 and TNBC cells secret the highest levels of IL-6 comparing to other subtypes of breast cancer. Therefore, IL-6 signaling represents a novel approach with a potential to improve the therapeutic efficacy. To date, however, no small molecules that target IL-6 signaling are available for clinical cancer therapy. IL-6 binds to IL-6 Rα, then recruits Glycoprotein 130 (GP130) to form hexamer consisting two IL-6/IL-6 Rα/GP130 heterotrimers to trigger a signaling cascade downstream including JAK/STAT3, PI3-K/AKT/mTOR, and MEK/ERK. Therefore, it is possible to target IL-6 signaling by blocking IL-6 binds to GP130 or IL-6 Rα and thus inhibiting its signaling cascade downstream. We have utilized a novel drug discovery approach combining Multiple Ligand Simultaneous Docking and drug repurposing to target GP130 and have identified a FDA-approved drug Bazedoxifene (for the prevention of the osteoporosis in postmenopausal women) with a novel function to inhibit IL-6 and GP130 proteinprotein interactions and thus blocking IL-6 signaling cascade downstream. In this study, we examined the therapeutic efficacy of Bazedoxifene in combination with paclitaxel on human TNBC cells and TNBC tumor model, aiming to provide experimental results of Bazedoxifene and paclitaxel combination for TNBC therapy. Our data showed Bazedoxifene inhibited increased STAT3 phosphorylation induced by IL-6. Bazedoxifene also inhibited phosphorylation of STAT3, AKT, and ERK in TNBC cells that produced IL-6. In addition, combination of Bazedoxifene with paclitaxel exhibited more significant inhibition on cell viability and colony formation and induction of apoptosis than single agent alone in TNBC cells in vitro. Moreover, Bazedoxifene inhibits cell migration and combination of Bazedoxifene with paclitaxel more significantly inhibit cell migration than single agent alone. We also showed bazedoxifene could inhibit increased proliferation of TNBC cells induced by IL-6. The combination of Bazedoxifene with paclitaxel also showed significantly inhibition of tumor growth of TNBC in vivo furtherly supported the therapeutic effects of Bazedoxifene. The results support the significant therapeutic efficacy of Bazedoxifene and paclitaxel combination for further TNBC therapy. Citation Format: Xiang Chen, Shengling Fu, Jilai Tian, Yang Cao, Chenglong Li, Jiayuh Lin. Repurposing FDA-approved drug bazedoxifene as a novel inhibitor of IL-6 signaling for triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 864.

Down-regulating IL-6/GP130 targets improved the anti-tumor effects of 5-fluorouracil in colon cancer.

Recent studies have confirmed that IL-6/GP130 targets are closely associated with tumor growth, metastasis and drug resistance. 5-Fluorouracil (5-FU) is the most common chemotherapeutic agent for colon cancer but is limited due to chemoresistance and high cytotoxicity. Bazedoxifene (BZA), a third-generation selective estrogen receptor modulator, was discovered by multiple ligand simultaneous docking and drug repositioning approaches to have a novel function as an IL-6/GP130 target inhibitor. Thus, we speculated that in colon cancer, the anti-tumor efficacy of 5-FU might be increased in combination with IL-6/GP130 inhibitors. CCK8 assay and colony formation assay were used to detect the cell proliferation and colony formation. We measured the IC50 value of 5-FU alone and in combination with BZA by cell viability inhibition. Cell migration and invasion ability were tested by scratch migration assays and transwell invasion assays. Flow cytometric analysis for cell apoptosis and cell cycle. Quantitative real-time PCR was used to detect Bad, Bcl-2 and Ki-67 mRNA expression and western blotting (WB) assay analyzed protein expression of Bad/Bcl-2 signaling pathway. Further mechanism study, WB analysis detected the key proteins level in IL-6/GP130 targets and JAK/STAT3, Ras/Raf/MEK/ERK, and PI3K/AKT/mTOR signaling pathway. A colon cancer xenograft model was used to further confirm the efficacy of 5-FU and BZA in vivo. The GP130, P-STAT3, P-AKT, and P-ERK expression levels were detected by immunohistochemistry in the xenograft tumor. BZA markedly potentiates the anti-tumor function of 5-FU in vitro and in vivo. Conversely, 5-FU activation is reduced following exogenous IL-6 treatment in cells. Further mechanistic studies determined that BZA treatment enhanced 5-FU anti-tumor activation by inhibiting the IL-6/GP130 signaling pathway and the phosphorylation status of the downstream effectors AKT, ERK and STAT3. In contrast, IL-6 can attenuate 5-FU function via activating IL-6R/GP130 signaling and the P-AKT, P-ERK and P-STAT3 levels. This study firstly verifies that targeting IL-6/GP130 signaling can increase the anti-tumor function of 5-FU; in addition, this strategy can sensitize cancer cell drug sensitivity, implying that blocking IL-6/GP130 targets can reverse chemoresistance. Therefore, combining 5-FU and IL-6/GP130 target inhibitors may be a promising approach for cancer treatment.

Abstract P5-14-02: Effects of bazedoxifene and/or letrozole in the prevention of chemically-induced mammary cancers

Abstract Bazedoxifene is described as a third-generation selective estrogen receptor modulator (SERM) that has been approved in Europe for the prevention and treatment of postmenopausal osteoporosis. The agent has also been shown to have therapeutic activity against estrogen receptor positive (ER+) breast cancer. Our laboratory has evaluated bazedoxifene for chemopreventive efficacy against mammary cancers when given alone or in combination with the aromatase inhibitor letrozole. Initially, the agents were evaluated alone to determine effective chemopreventive doses in the methylnitrosourea (MNU) ER+ mammary cancer model using female Sprague-Dawley rats. Bazedoxifene was mixed directly into a standard (Teklad, 4% fat) diet, while letrozole was gavaged 7x/week (vehicle was ethanol: PEG 400, 10:90, v/v). When evaluated alone in rats receiving MNU at 50 days of age, bazedoxifene (started one week after MNU) at doses of 100 and 30 mg/kg diet decreased mammary cancer multiplicity by 93 and 88%, respectively. In a similar protocol, letrozole at a dose of 0.1 mg/kg BW/day reduced cancer multiplicity by 89%, while a dose of 0.05 mg/kg BW/day reduced the number by 47%. In a separate study evaluating lower doses of bazedoxifene (5 mg/kg diet) and letrozole (0.04 mg/kg BW/day), mammary cancer multiplicity was decreased by 73% by both agents. When the agents were given in combination at these lower dose levels, cancer number was reduced by 91%. It is obvious that bazedoxifene is as effective as letrozole in the prevention of ER+ mammary cancers. Furthermore, administration of the agents in combination suggests an additive effect. The large increase in body weight gain generally noted in letrozole treated rats was not observed in rats receiving bazedoxifene; of interest, rats receiving the combination also had no increase in body weights. Several published invitro studies suggested that bazedoxifene might also be effective in preventing ER- mammary cancers; e.g., the agent is a novel inhibitor of IL-6/GP130 protein-protein interactions using multiple ligand simultaneous docking and drug repositioning approaches that result in apoptosis. In an initial study using MMTV/Neu mice (N=21/group) that received dimethylbenzanthracene (DMBA), we showed that administering bazedoxifene (15 mg/kg diet) would reduce the mammary tumor multiplicity from 5.4/mouse in controls to 1.1/mouse (an 80% decrease). These studies suggested that bazedozxifene is superior to other similar agents in that both ER+ and ER- mammary cancers are prevented at non-toxic doses. Supported by NCI contract HHSN261201200021I, Task Order HHSN26100007. Citation Format: Dunn BK, Grubbs CJ. Effects of bazedoxifene and/or letrozole in the prevention of chemically-induced mammary cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-14-02.

Abstract 186: FDA approved drug Bazedoxifene as a novel inhibitor of IL 6 and IL 11/GP130 signaling for osteosarcoma therapy

Abstract IL-6 and L-11 signaling pathways play an important role in cancer cell survival and progression, including osteosarcoma. IL-6/IL-11 binds to IL-6/IL-11 Rα to form a binary complex, then recruits GP130 to form the IL-6/IL-6 Rα/GP130 or IL-11/IL-11 Rα/GP130 heterotrimer and triggers a signaling cascade downstream. One of the major downstream effectors of IL-6 is STAT3. STAT3 is persistently activated in many human osteosarcoma specimens and cell lines and STAT3 is required for osteosarcoma cells survival. So IL-6 and IL-11 present a viable novel target for osteosarcoma therapy. To date, however, no small molecules that target both IL-6/GP130 and IL-11/GP130 signaling pathways are available for cancer therapy. To overcome this critical problem, we have utilized a novel drug discovery approach combining Multiple Ligand Simultaneous Docking and drug repositioning to target GP130. Drug repositioning refers to reuse fragments from the existing FDA-approved drugs for new applications and could potentially reposition the existing drugs as novel, off-target inhibitors of the GP130 as their new target. Using this novel method, we have identified a FDA-approved drug Bazedoxifene with a novel function to inhibit IL-6 and GP130 proteinprotein interactions. Furthmore, because IL-11 binds to the same domain of GP130 as IL-6, Bazedoxifene should also inhibit IL-11/GP130 signaling. Accordingly, Bazedoxifene can indeed inhibit the induction of P-STAT3 by both IL-6 and IL-11. Bazedoxifene appeared specific to IL-6 and IL-11 as phosphorylation of STAT1 and STAT3 by IFN-γ and LIF respectively were not affected by the compound. Bazedoxifene inhibited P-STAT3 and induced apoptosis in human osteosarcoma cell lines expressing IL-6 and IL-11. In addition, Bazedoxifene can inhibit colony formation after the drug treatments in cancer cells.IL-6 but not IFN-γ could rescue the Bazedoxifene-mediated inhibition of cell viability in osteosarcoma cells. These results further support that IL-6/GP130 signaling pathway is one of the main targets of Bazedoxifene-mediated inhibition in osteosarcoma cells. To determine the in vivo activity of Bazedoxifene, we further tested the efficacy of Bazedoxifene in tumor xenografts generated from the SJSA osteosarcoma cells that show persistent IL-6/STAT3 activation. Bazedoxifene via oral gavage inhibited P-STAT3 and the growth of SJSA tumor xenografts. These data demonstrated that Bazedoxifene is potent in suppressing tumor growth and is orally bioavailable in inhibiting P-STAT3. It further indicates that Bazedoxifene is a promising IL-6/GP130-targeting drug, which likely to have in vivo anti-tumor activity in osteosarcoma. In summary, Bazedoxifene already approved for safety by the FDA as a novel inhibitor of IL-6/GP130 and IL-11/GP130 signaling should provide an easier path to clinical trials and have a potential to improve the outcome of osteosarcoma. Note: This abstract was not presented at the meeting. Citation Format: Xiaojuan Wu, Hui Xiao, Chenglong Li, Jiayuh Lin. FDA approved drug Bazedoxifene as a novel inhibitor of IL 6 and IL 11/GP130 signaling for osteosarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 186. doi:10.1158/1538-7445.AM2017-186