Ligand-Based Pharmacophore Modeling, Molecular Docking, and Molecular Dynamic Studies of Dual Tyrosine Kinase Inhibitor of EGFR and VEGFR2.

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) play an important role in cancer growth. Both of them have close relationships. Expression of EGFR will induce an angiogenic factor (VEGF) release for binding with VEGFR2. However, the existence of VEGF up-regulation independent of EGFR leads to cancer cell resistance to anti-EGFR. Therefore, a therapeutic approach targeting EGFR and VEGFR2 simultaneously may improve the outcome of cancer treatment. The present study was designed to identify potential compounds as a dual inhibitor of EGFR and VEGFR2 by the computational method. Firstly, the ligand-based pharmacophore model for each target was setup to screen of ZINC database of purchasable compounds. The hit compounds obtained by pharmacophore screening were then further screened by molecular docking studies. Taking erlotinib (EGFR inhibitor) and axitinib (VEGFR2 inhibitor) as reference drugs, six potential compounds (ZINC08398597, ZINC12047553, ZINC16525481, ZINC17418102, ZINC21942954, and ZINC38484632) were selected based on their docking scores and binding interaction. However, molecular dynamics simulations demonstrated that only ZINC16525481 and ZINC38484632 which have good binding free energy and stable hydrogen bonding interactions with EGFR and VEGFR2. The result represents a promising starting point for developing potent dual tyrosine kinases inhibitor of EGFR and VEGFR2.

Identification and Validation of VEGFR2 Kinase as a Target of Voacangine by a Systematic Combination of DARTS and MSI.

Although natural products are an important source of drugs and drug leads, identification and validation of their target proteins have proven difficult. Here, we report the development of a systematic strategy for target identification and validation employing drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) without modifying or labeling natural compounds. Through a validation step using curcumin, which targets aminopeptidase N (APN), we successfully standardized the systematic strategy. Using label-free voacangine, an antiangiogenic alkaloid molecule as the model natural compound, DARTS analysis revealed vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein. Voacangine inhibits VEGFR2 kinase activity and its downstream signaling by binding to the kinase domain of VEGFR2, as was revealed by docking simulation. Through cell culture assays, voacangine was found to inhibit the growth of glioblastoma cells expressing high levels of VEGFR2. Specific localization of voacangine to tumor compartments in a glioblastoma xenograft mouse was revealed by MSI analysis. The overlap of histological images with the MSI signals for voacangine was intense in the tumor regions and showed colocalization of voacangine and VEGFR2 in the tumor tissues by immunofluorescence analysis of VEGFR2. The strategy employing DARTS and MSI to identify and validate the targets of a natural compound as demonstrated for voacangine in this study is expected to streamline the general approach of drug discovery and validation using other biomolecules including natural products.

Andrographolide binds to ATP-binding pocket of VEGFR2 to impede VEGFA-mediated tumor-angiogenesis.

Vasculogenesis and angiogenesis are process of formation of blood vessels. Blood vessels are evolved to distribute nutrients and oxygen to distant organs. These vessels are crucial for growth and repair of wounded tissue. During tumor condition there occurs imbalance in the growth of blood vessels which leads to neo-angiogenesis. Neo-angiogenesis is major perpetrator behind the establishment of tumor. Tumor cells secrete pro-angiogenic factor VEGFA which binds to VEGFR2 present over surface of endothelial cells and triggers formation of new blood vessels. To inhibit tumor-angiogenesis, a physiologically-safe small molecule inhibitor was screened which can potentially interact with kinase domain of VEGFR2 and inhibit its activity. Molecular-docking module and biochemical analysis identified andrographolide as one of the best docking molecules that binds to ATP-binding pocket of VEGFR2 and inhibits its kinase activity. Thus, for a more radical approach towards safe VEGFR2 inhibitor, andrographolide was repurposed to inhibit tumor-angiogenesis and reduce tumor burden.

Structure-based identification of potent VEGFR-2 inhibitors from in vivo metabolites of a herbal ingredient.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is one of the regulatory elements of angiogenesis that is expressed highly in various diseases and is also essential for solid tumor growth. The present study was aimed at identifying potent inhibitors of VEGFR-2 by considering herbal secondary metabolites; as natural molecules are less toxic than synthetic derivatives. A structure-based virtual screening protocol consisting of molecular docking, MM-GBSA and ADME/T analysis was initially used to screen a library of in vivo metabolites of the herbal ingredient. Using a fixed cutoff value, four potent virtual hits were identified from molecular docking, ADME/T and binding affinity calculations, which were considered further for molecular dynamics (MD) simulation to broadly describe the binding mechanisms to VEGFR-2. The results suggested that these molecules have high affinity for the catalytic region of VEGFR-2, and form strong hydrophobic and polar interactions with the amino acids involved in the binding site of ATP and linker regions of the catalytic site. Subsequently, the stability of the docked complexes and binding mechanisms were evaluated by MD simulations, and the energy of binding was calculated through MM-PBSA analysis. The results uncovered two virtual hits, designated ZINC14762520 and ZINC36470466, as VEGFR-2 inhibitors, and suggested that they bind to kinase domain in an ATP-competitive manner. These virtual hits will offer a suitable starting point for the further design of their various analogs, allowing a rational search for more effective inhibitors in the future. Graphical abstract.

Anti-angiogenic Activity of Major Phenolics in Tamarind Assessed with Molecular Docking Study on VEGF Kinase Proteins.

BACKGROUND AND OBJECTIVE: The waste products of the tamarind canning industry have been discarded; however, it has potential health benefits. Herein, the study was carried out HPLC profiling of phenolic constituents of Tamarindis indica pericarp and seeds. Furthermore, the cytotoxic activity against HUH-7 cells was evaluated and assessed with molecular docking study on angiogenesis-related VEGF kinase proteins in addition to evaluating the level of released VEGF in treated HUH-7 cells by ELISA. MATERIALS AND METHODS: Folin-ciocalteu and AlCl3 assays were used for quantification of total phenolics (TPC) and total flavonoids (TFC) contents, respectively. Molecular docking study was done on VEGF kinase proteins. RESULTS: TPC and TFC of pericarp and seeds were 0.35±0.02 g GAE g-1 DE and 0.12±0.009 g CE g-1 DE, 0.39±0.01 g GAE g-1 DE and 0.03±0.006 g CE g-1 DE, respectively. In pericarp, 8 phenolics were tentatively identified, where (+)-catechin was the major (27,386.04 µg g-1 DE) followed by gallic acid and naringenin (931.47, 500.42 µg g-1 DE) respectively. While in seeds, 11 phenolics were tentatively identified, where naringenin was the major (95,305.47 µg g-1 DE) followed by (+)-catechin and rutin (54,930.29, 15,361.66 µg g-1 DE) respectively. Aqueous and methanol seeds extracts exhibit cytotoxic effect with IC50 27.4±1.81 and 13.4±0.94 µg mL-1, respectively, it was more potent than aqueous and methanol pericarp extracts which had IC50 132±5.82 and 61.6±3.16 µg mL-1. The tested phenolics were fit on the active sites of VEGF kinase targets with varied degree of interactions. The cytotoxic and anti-angiogenic activities were confirmed in light of phenolics docking interactions. CONCLUSION: Results demonstrate for the first time that phenolics could inhibit angiogenesis via inhibiting kinase proteins, which could therefore be developed as antiangiogenic drugs.

Discovery of Novel Pazopanib-Based HDAC and VEGFR Dual Inhibitors Targeting Cancer Epigenetics and Angiogenesis Simultaneously.

Herein a novel series of pazopanib hybrids as polypharmacological antitumor agents were developed based on the crosstalk between histone deacetylases (HDACs) and vascular endothelial growth factor (VEGF) pathway. Among them, one ortho-aminoanilide 6d and one hydroxamic acid 13f exhibited considerable total HDACs and VEGFR-2 inhibitory activities. The HDAC inhibitory activities endowed 6d and 13f with potent antiproliferative activities, which was not observed in the approved VEGFR inhibitor pazopanib. Compounds 6d and 13f possessed comparable HDAC isoform selectivity profiles to the clinical class I HDAC inhibitor MS-275 and the approved pan-HDAC inhibitor SAHA, respectively. 6d and 13f also exhibited uncompromised multiple tyrosine kinases inhibitory activities relative to pazopanib. The intracellular dual inhibition to HDAC and VEGFR of 6d and 13f was validated by Western blot analysis. In both HUVECs tube formation assay and rat thoracic aorta rings assay, 6d and 13f showed comparable antiangiogenic potencies to pazopanib. What's more, 6d possessed desirable pharmacokinetic profiles with the oral bioavailability of 72% in SD rats and considerable in vivo antitumor efficacy in a human colorectal adenocarcinoma (HT-29) xenograft model.

Penduliflaworosin, a Diterpenoid from Croton crassifolius, Exerts Anti-Angiogenic Effect via VEGF Receptor-2 Signaling Pathway.

Anti-angiogenesis targeting vascular endothelial growth factor receptor-2 (VEGFR-2) has been considered as an important strategy for cancer therapy. Penduliflaworosin is a diterpenoid isolated from the plant Croton crassifolius. Our previous study showed that this diterpenoid possesses strong anti-angiogenic activity by inhibiting vessel formation in zebrafish. This study was conducted to further investigate the anti-angiogenic activity and mechanism of penduliflaworosin. Results revealed that penduliflaworosin significantly inhibited VEGF-induced angiogenesis processes including proliferation, invasion, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Moreover, it notably inhibited VEGF-induced sprout formation of aortic rings and blocked VEGF-induced vessel formation in mice. Western blotting studies showed that penduliflaworosin inhibited phosphorylation of the VEGF receptor-2 and its downstream signaling mediators in HUVECs, suggesting that the anti-angiogenic activity was due to an interference with the VEGF/VEGF receptor-2 pathway. In addition, molecular docking simulation indicated that penduliflaworosin could form hydrogen bonds within the ATP-binding region of the VEGF receptor-2 kinase unit. Finally, cytotoxicity assay showed that penduliflaworosin possessed little toxicity toward both cancer and normal cells. Taken together, our findings demonstrate that penduliflaworosin exerts its anti-angiogenic effect via the VEGF receptor-2 signaling pathway. The anti-angiogenic property and low cytotoxicity of penduliflaworosin suggest that it may be useful in cancer treatments.

Strong Anti-tumorous Potential of Nardostachys jatamansi Rhizome Extract on Glioblastoma and In Silico Analysis of its Molecular Drug Targets.

BACKGROUND: Glioblastoma has been reckoned as the prime cause of death due to brain tumours, being the most invasive and lethal. Available treatment options, i.e. surgery, radiotherapy, chemotherapy and targeted therapies are not effective in improving prognosis, so an alternate therapy is insistent. Plant based drugs are efficient due to their synergistic action, multi-targeted approach and least side effects. METHODS: The anti-tumorous potential of Nardostachys jatamansi rhizome extract (NJRE) on U87 MG cell line was evaluated through various in vitro and in silico bio-analytical tools. RESULTS: NJRE had a strong anti-proliferative effect on U87 MG cells, Its IC50 was 33.73±3.5, 30.59±3.4 and 28.39±2.9 µg/mL, respectively after 24, 48 and 72 h. NJRE at 30 µg/mL induced DNA fragmentation, indicating apoptosis, early apoptosis began in the cells at 20 µg/mL, whereas higher doses exhibited late apoptosis as revealed by dual fluorescence staining. NJRE at 60 and 80 µg /mL caused a G0/G1 arrest and at 20 and 40 µg/mL showed excessive nucleation and mitotic catastrophe in the cells. Immuno-blotting validated the apoptotic mode of cell death through intrinsic pathway. NJRE was harmless to normal cells. In silico docking of NJRE marker compounds: oroselol, jatamansinol, nardostachysin, jatamansinone and nardosinone have revealed their synergistic and multi-targeted interactions with Vestigial endothelial growth factor receptor 2 (VEGFR2), Cyclin dependent kinase 2 (CDK2), B-cell lymphoma 2 (BCL2) and Epidermal growth factor receptor (EGFR). CONCLUSION: A strong dose specific and time dependent anti-tumorous potential of NJRE on U87 MG cells was seen. The extract can be used for the development of safe and multi-targeted therapy to manage glioblastoma, which has not been reported earlier.

Development of APTES-Decorated HepG2 Cancer Stem Cell Membrane Chromatography for Screening Active Components from Salvia miltiorrhiza.

Cell membrane chromatography (CMC) is an ideal method for screening potential active components acting on target cell membranes from a complex system, such as herbal medicines. But due to the decay and falling-off of membranes, the CMC column suffers from short life span and low reproducibility. This has greatly limited the application of this model, especially when the cell materials are hard to obtain. To solve this problem, a novel type of (3-aminopropyl)triethoxysilane (APTES)-decorated silica gel was employed. The silica gel was decorated with aldehydes with the help of APTES, which react with the amino groups on cell membranes to form a covalent bond. In this way, cell membranes were immobilized on the surface of silica gel, so it is not easy for membranes to fall off. According to our investigation, the column life of the APTES-decorated group was prolonged to more than 12 days, while the control group showed a sharp decline in column efficiency in the first 3 days. To verify this model, a novel APTES-decorated HepG2 cancer stem cell membrane chromatography (CSCMC) was established and applied in a comprehensive two-dimensional chromatographic system to screen potential active components in Salvia miltiorrhiza. As a result, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I were retained on this model and proved to be effective on HepG2 cancer stem cells by the following cell proliferation and apoptosis assay, with IC50 of 10.30 µM, 17.85 µM, and 2.53 µM, respectively. This improvement of CMC can significantly prolong its column life span and broaden the range of its application, which is very suitable for making invaluable or hard-to-obtain cell materials, such as stem cells, for specific drug screening.

Recent advances in structure-based drug design and virtual screening of VEGFR tyrosine kinase inhibitors.

During the past decade, developments in computational processing and X-ray crystallography have allowed virtual screening become integrated into drug discovery campaigns. This review focuses on the recent advancements in the drug discovery of VEGFR2 tyrosine kinase inhibitors (VEGFR2 TKIs) by using in silico methodologies. An introduction for the methodology framework of pharmacophore modeling, molecular docking and structure-based design are provided. We discuss the recent studies on the structures of VEGFR2 protein kinase in different binding modes, and the insights on molecular interactions gained from knowledge of the co-crystal structures complex with structurally diverse VEGFR2 inhibitors. We provide some aspects of model construction and molecular docking techniques. Several representative examples of successful applications on VEGFR2 virtual screening for hit discovery, lead optimization and structure-based design are also presented.

Evaluation of the impact of transient interruption of antiangiogenic treatment using ultrasound-based techniques in a murine model of hepatocellular carcinoma.

BACKGROUND: Development of escape pathways from antiangiogenic treatments was reported to be associated with enhanced tumor aggressiveness and rebound effect was suggested after treatment stop. Aim of the study was to evaluate tumor response simulating different conditions of administration of antiangiogenic treatment (transient or definitive treatment stop) in a mouse model of hepatocellular carcinoma. METHODS: Subcutaneous tumors were created by inoculating 5 × 10(6) Huh7 cells into the right flank of 14 nude mice. When tumor size reached 5-10 mm, mice were divided in 3 groups: group 1 was treated with placebo, group 2 was treated with sorafenib (62 mg/kg via gavage) but temporarily suspended from day +5 to +9, whereas in group 3 sorafenib was definitively stopped at day +5. At day +13 all mice were sacrificed, collecting masses for Western-Blot analyses. Volume was calculated with B-mode ultrasonography at day 0, +5, +9, +11 and +13. VEGFR2-targeted contrast-enhanced ultrasound using BR55 (Bracco Imaging) was performed at day +5 and +13 and elastonosography (Esaote) at day +9 and +11 to assess tumor stiffness. RESULTS: Median growth percentage delta at day +13 versus day 0 was 197% (115-329) in group 1, 81% (48-144) in group 2 and 111% (27-167) in group 3. Median growth delta at day +13 with respect to day +5 was 79% (48-127), 37% (-14128) and 81% (15-87) in groups 1, 2 and 3, respectively. Quantification of targeted-CEUS at day +13 showed higher values in group 3 (509 Arbitrary Units AI, range 293-652) than group 1 (275 AI, range 191-494) and group 2 (181 AI, range 63-318) (p=0.033). Western-Blot analysis demonstrated higher VEGFR2 expression in group 3 with respect to group 1 and 2. CONCLUSIONS: A transient interruption of antiangiogenic treatment does not impede restoration of tumor response, while a definitive interruption tends to stimulate a rebound of angiogenesis to higher level than without treatment.

Dietary compound isoliquiritigenin inhibits breast cancer neoangiogenesis via VEGF/VEGFR-2 signaling pathway.

Angiogenesis is crucial for cancer initiation, development and metastasis. Identifying natural botanicals targeting angiogenesis has been paid much attention for drug discovery in recent years, with the advantage of increased safety. Isoliquiritigenin (ISL) is a dietary chalcone-type flavonoid with various anti-cancer activities. However, little is known about the anti-angiogenic activity of isoliquiritigenin and its underlying mechanisms. Herein, we found that ISL significantly inhibited the VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) at non-toxic concentration. A series of angiogenesis processes including tube formation, invasion and migration abilities of HUVECs were also interrupted by ISL in vitro. Furthermore, ISL suppressed sprout formation from VEGF-treated aortic rings in an ex-vivo model. Molecular mechanisms study demonstrated that ISL could significantly inhibit VEGF expression in breast cancer cells via promoting HIF-1α (Hypoxia inducible factor-1α) proteasome degradation and directly interacted with VEGFR-2 to block its kinase activity. In vivo studies further showed that ISL administration could inhibit breast cancer growth and neoangiogenesis accompanying with suppressed VEGF/VEGFR-2 signaling, elevated apoptosis ratio and little toxicity effects. Molecular docking simulation indicated that ISL could stably form hydrogen bonds and aromatic interactions within the ATP-binding region of VEGFR-2. Taken together, our study shed light on the potential application of ISL as a novel natural inhibitor for cancer angiogenesis via the VEGF/VEGFR-2 pathway. Future studies of ISL for chemoprevention or chemosensitization against breast cancer are thus warranted.

Discovering small molecule ligands of vascular endothelial growth factor that block VEGF-KDR binding using label-free microarray-based assays.

We present here a label-free microarray-based assay platform that we used to identify inhibitors of vascular endothelial growth factor (VEGF)-kinase-insertion domain receptor (KDR) binding. Supported by a combination of special ellipsometry-based optical detection and small molecule microarrays (SMM), this platform consists of three assays: (1) the first assay detects binding of a target protein with SMM and identifies ligands to the protein as inhibitor candidates; (2) the second assay detects binding of a receptor protein with identical SMM and subsequent binding of the target protein (a sandwich assay) to identify the ligands to the receptor protein that do not interfere with the target-receptor binding; (3) the third assay detects binding of the target protein to the receptor protein in the presence of the ligands of the target protein identified from the first assay, with the receptor protein immobilized to a solid surface through the ligands identified in the second assay, to yield dose-response curves. Using this platform, we screened 7,961 compounds from the National Cancer Institute and found 12 inhibitors to VEGF-KDR (VEGFR2) interactions with IC50 ranging from 0.3 to 60 µM. The inhibitory potency of these inhibitors found in the microarray-based assay was confirmed by their inhibition of VEGF-induced VEGFR2 phosphorylation in a cell-based assay.

Synthesis and in vitro evaluation of [18F](R)-FEPAQ: a potential PET ligand for VEGFR2.

Synthesis and in vitro evaluation of [(18)F](R)-N-(4-bromo-2-fluorophenyl)-7-((1-(2-fluoroethyl)piperidin-3-yl)methoxy)-6-methoxyquinazolin-4-amine ((R)-[(18)F]FEPAQ or [(18)F]1), a potential imaging agent for the VEGFR2, using phosphor image autoradiography are described. Synthesis of 2, the desfluoroethyl precursor for (R)-FEPAQ was achieved from t-butyl 3-(hydroxymethyl)piperidine-1-carboxylate (3) in five steps and in 50% yield. [(18)F]1 was synthesized by reaction of sodium salt of compound 2 with [(18)F]fluoroethyl tosylate in DMSO. The yield of [(18)F]1 was 20% (EOS based on [(18)F]F(-)) with >99% radiochemical purity and specific activity of 1-2 Ci/µmol (n=10). The total synthesis time was 75 min. The radiotracer selectively labeled VEGFR2 in slide-mounted sections of human brain and higher binding was found in surgically removed human glioblastoma sections as demonstrated by in vitro phosphor imager studies. These findings suggest [(18)F]1 may be a promising radiotracer for imaging VEGFR2 in brain using PET.

Synthesis and biological profile of the pan-vascular endothelial growth factor receptor/tyrosine kinase with immunoglobulin and epidermal growth factor-like homology domains 2 (VEGF-R/TIE-2) inhibitor 11-(2-methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (CEP-11981): a novel oncology therapeutic agent.

A substantial body of evidence supports the utility of antiangiogenesis inhibitors as a strategy to block or attenuate tumor-induced angiogenesis and inhibition of primary and metastatic tumor growth in a variety of solid and hematopoietic tumors. Given the requirement of tumors for different cytokine and growth factors at distinct stages of their growth and dissemination, optimal antiangiogenic therapy necessitates inhibition of multiple, complementary, and nonredundant angiogenic targets. 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11b, CEP-11981) is a potent orally active inhibitor of multiple targets (TIE-2, VEGF-R1, 2, and 3, and FGF-R1) having essential and nonredundant roles in tumor angiogenesis and vascular maintenance. Outlined in this article are the design strategy, synthesis, and biochemical and pharmacological profile for 11b, which completed Phase I clinical assessing safety and pharmacokinetics allowing for the initiation of proof of concept studies.

Scaffold oriented synthesis. Part 1: Design, preparation, and biological evaluation of thienopyrazoles as kinase inhibitors.

We report the synthesis of kinase targeted libraries based on the thienopyrazole scaffold. Several thienopyrazole analogs have been identified as submicromolar inhibitors of KDR.