Targeting Vascular Endothelial Growth Factor Receptors Research Articles

PyPLIF HIPPOS-aided Construction and Retrospective Validation of Structure-Based Virtual Screening Protocol Targeting VEGFR2

Recently, the discovery of small molecules as inhibitors for vascular endothelial growth factor receptor 2 (VEGFR2) is of timely interest, especially in the area of ocular neovascular diseases. On the other hand, PyPLIF HIPPOS in combination with machine learning techniques has been reported to increase the prediction quality of structure-based virtual screening (SBVS) protocols. The original version of PyPLIF has served in the development of an SBVS protocol that successfully identified novel chalcone derivatives and short peptides as potent inhibitors for acetylcholinesterase. In this short communication, construction and retrospective validation of an SBVS protocol employing PyPLIF HIPPOS targeting VEGFR2 are presented to make it publicly available. The retrospective validation employed 409 active compounds and 24,950 decoys from the enhanced version of the directory of useful decoys. All compounds were docked independently 3 times using AutoDock Vina followed by the identification of the protein-ligand interaction fingerprints (PLIF) employing PyPLIF HIPPOS. The derived ensemble PLIF descriptors were then used in the decision tree construction using a machine-learning technique called recursive partitioning and regression trees. The best decision was then incorporated in the SBVS protocol. The F-measure and enrichment factor values of the SBVS protocol were 0.387 and 76.879, respectively. Hence, the SBVS protocol is readily available to screen small molecules or short peptides.

Molecular Docking and Dynamic Simulation of Erythrina fusca Lour Chemical Compounds Targeting VEGFR-2 Receptor for Anti-Liver Cancer Activity

Liver cancer is a serious health concern characterized by abnormal cell growth, but currently, available treatment options are limited, suggesting the need for a new therapeutic method. Therefore, this research aimed to investigate the potential of chemical compounds obtained from the cangkring plant (Erythrina fusca) as anti-liver cancer agents targeting Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2). The investigation was conducted in silico through molecular docking and dynamic method. Molecular docking was performed using AutoDock Tools, followed by visualization with Biovia Discovery Studio. Additionally, molecular dynamics simulation was conducted using GROMACS software and visualized with Grace. A total of 36 chemical compounds from E. fusca were used as ligands for molecular docking. The results showed that Isobavachalcone (ISB) was the most effective test compound with a binding energy of -11.45 kcal/mol, compared to the positive control Sorafenib with a value of - 11.51 kcal/mol. In this context, hydrogen bonding, as well as hydrophobic, electrostatic, and unfavorable molecular interactions were identified. Moreover, molecular dynamics simulation provided RMSD, RMSF, Radius of Gyration (Rg), and hydrogen bond parameters. Analysis of these parameters further confirmed the superior stability of ISB in binding to VEGFR-2, suggesting the potential to suppress angiogenesis by blocking the receptor.

Development and assessment of novel pyrazole-thiadiazol hybrid derivatives as VEGFR-2 inhibitors: design, synthesis, anticancer activity evaluation, molecular docking, and molecular dynamics simulation.

Cancer remains a significant health challenge globally, requiring the development of targeted chemotherapeutics capable of specifically inhibiting cancer cell growth. Angiogenesis is one of the key features of tumor growth and metastasis and is, therefore, an important target for the treatment of many tumors. The vascular endothelial growth factor (VEGF) signaling pathway has proven to be a promising lead in anticancer therapy due to the central role it plays in tumor angiogenesis. Vascular endothelial growth factor receptor-2 (VEGFR-2) is a key mediator in the signaling pathway regulating angiogenesis. Targeting VEGFR-2 may disrupt angiogenesis, leading to a reduction in tumor blood supply and tumor progression. The design, synthesis, and assessment of novel VEGFR-2 inhibitor derivatives are the focus of this study, with particular emphasis on incorporating the pyrazole-thiadiazol pharmacophore into the molecular structure. Taking advantage of the pharmacophoric properties of pyrazole and 1,3,4-thiadiazol, compounds with different substituents in the main structure were designed and synthesized. The compounds were also evaluated for antiproliferative activity against cancer cell lines. Compound 4e demonstrated the highest activity among all compounds, with an IC50 of 9.673±0.399 μM against HT-29 cells and 23.081±0.400 μM against NIH3T3 cells. To further support the inhibitory activity of compound 4e, an in silico study was performed. Compound 4e demonstrated strong binding to the active site of VEGFR-2 in molecular docking studies, forming hydrogen bonds with key amino acid residues. The stability of the compound in the enzyme's active site was demonstrated through molecular dynamics simulations.

Integrated in silico and in vitro discovery of a new anticancer thiadiazole analog targeting VEGFR-2

In this work, a new thiadiazole analogue (TDA) was designed with the intention of incorporating key features of Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) inhibitors, and then was examined computationally and experimentally. In vitro (cytotoxicity on MCF-7, HepG2 and WI-38 cell lines, in vitro VEGFR-2 assay, cell cycle analysis, apoptosis assays, RT-PCR, and migration studies), in addition to in silico (molecular docking, ADMET, MD simulations and DFT studies) were conducted. Prior to synthesis, docking and ADMET studies yielded predictions indicating favorable activity and drug likeness. After synthesis, the results demonstrated promising inhibitory activity of TDA on MCF-7 and HepG2 cancer cell lines. These results were notably close to those of sorafenib. The in vitro VEGFR-2 assay revealed significant inhibition, with an IC50 value of 0.283 µM. Selectivity studies indicated a favorable safety profile, with reduced toxicity towards normal lung cells (WI-38). Cell cycle analysis of TDA revealed a significant arrest in the G0-G1 phase, compared to the control group. Apoptosis assays demonstrated a considerable increase in programmed cell death, particularly in the early stages as opposed to the control group. Additionally, there was a notable increase in the expression of the pro-apoptotic gene Bax and a downregulation of the anti-apoptotic gene Bcl2 in MCF-7 cells treated with TDA. TDA also exhibited an anti-angiogenic potential through decreasing the abilities of the MCF-7 cells to migrate and heal. Computationally, various MD simulations studies indicated stable interactions between TDA and VEGFR-2 over 200 ns on both mechanical and energetic levels. DFT studies confirmed reactivity and stability of TDA. These comprehensive findings emphasize TDA’s potential as a multi-targeted candidate for further development in cancer therapy. The promising inhibitory activity, favorable safety profile, and mechanistic insights highlight TDA as a noteworthy contender through further in vivo studies and modifications.

Understanding Sorafenib-Induced Cardiovascular Toxicity: Mechanisms and Treatment Implications.

Tyrosine kinase inhibitors (TKIs) have been recognized as crucial agents for treating various tumors, and one of their key targets is the intracellular site of the vascular endothelial growth factor receptor (VEGFR). While TKIs have demonstrated their effectiveness in solid tumor patients and increased life expectancy, they can also lead to adverse cardiovascular effects including hypertension, thromboembolism, cardiac ischemia, and left ventricular dysfunction. Among the TKIs, sorafenib was the first approved agent and it exerts anti-tumor effects on hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) by inhibiting angiogenesis and tumor cell proliferation through targeting VEGFR and RAF. Unfortunately, the adverse cardiovascular effects caused by sorafenib not only affect solid tumor patients but also limit its application in curing other diseases. This review explores the mechanisms underlying sorafenib-induced cardiovascular adverse effects, including endothelial dysfunction, mitochondrial dysfunction, endoplasmic reticulum stress, dysregulated autophagy, and ferroptosis. It also discusses potential treatment strategies, such as antioxidants and renin-angiotensin system inhibitors, and highlights the association between sorafenib-induced hypertension and treatment efficacy in cancer patients. Furthermore, emerging research suggests a link between sorafenib-induced glycolysis, drug resistance, and cardiovascular toxicity, necessitating further investigation. Overall, understanding these mechanisms is crucial for optimizing sorafenib therapy and minimizing cardiovascular risks in cancer patients.

Targeting EGFR and VEGFR-2 Kinases With Nanoparticles: A Computational Approach for Cancer Therapy Advancement

The study investigates titanium and zinc nanoparticles as inhibitors for the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2), pivotal regulators of cell processes. VEGFR-2 activation fuels tumor angiogenesis in cancer cells, sustaining malignant tissue expansion. Molecular docking analysis illustrates the nanoparticles’ binding to the active sites, inhibiting the phosphorylation of key proteins in downstream signaling. This inhibition offers a promising therapeutic approach to impede cancer-related signaling, potentially slowing down aberrant protein cascades controlled by EGFR and VEGFR-2. The findings propose a novel avenue for cancer treatment, targeting abnormal growth pathways using titanium and zinc nanoparticles.

Chimeric antigen receptor macrophages activated through TLR4 or IFN-γ receptors suppress breast cancer growth by targeting VEGFR2.

Chimeric antigen receptor macrophage (CAR-M) is a promising immunotherapy strategy of anti-tumor due to its high infiltration, direct phagocytosis of tumor cells, immunomodulation of tumor microenvironment (TME) and linkage of innate and adaptive immunity. Here a series of novelly designed CAR-Ms by targeting vascular endothelial growth factor receptor-2 (VEGFR2), which highly expressed in tumor cells and TME, were evaluated. Their activation signals were transduced by Tlr4 or Ifn-γ receptors either alone or in combination, which were designed to mediate M1 polarization of macrophages as the downstream of lipopolysaccharide or Ifn-γ that had been widely reported. Our results showed that VEGFR2-targeting CAR-Ms could be activated under the stimulation of VEGFR2-expressing cells. They exhibited higher expression of CD86, MHCII and TNF-α in vitro and enhanced tumor suppressive abilities in vivo. Implantation of these CAR-Ms into 4T1 breast cancer-bearing mice could obviously inhibit the progression of tumor without significant toxic side effects, especially the group of mmC in which constructed with Tlr4 as the intracellular domain of CAR. In conclusion, this research provides a promising design of CAR that induce macrophages activation by Tlr4 and/or Ifn-γ receptors, and these CAR-Ms could effectively inhibit tumor growth through targeting VEGFR2.

Preclinical Evaluation of Novel Tyrosine-Kinase Inhibitors in Medullary Thyroid Cancer.

Simple SummaryMedullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular calcitonin-secreting C cells of the thyroid. Most of the patients affected by MTC, especially the familial form, harbor a mutation of the RET proto-oncogene. In patients with advanced disease, medical therapy is represented by two tyrosine-kinase inhibitors: cabozantinib and vandetanib. However, their usage is limited by several adverse events and drug-resistance onset. The aim of this preclinical study was to evaluate the antitumor activity of novel molecules for the therapy of MTC: SU5402, an inhibitor of the fibroblast growth factor receptor type 1 (FGFR-1) and vascular endothelial growth factor receptor (VEGFR)-2; sulfatinib, a multi-target kinase inhibitor selective for FGFR-1 and the VEGFR-1, -2, and -3; SPP86, a RET-specific inhibitor. Our results suggest a potential role in targeting the FGFR and VEGFR signaling pathways as an alternative strategy for resistant tumors and a significative antitumor activity of this new RET-specific inhibitor.Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular C cells of the thyroid gland. In this preclinical study, we tested three tyrosine-kinase inhibitors (TKIs): SU5402, a selective inhibitor of fibroblast growth factor receptor (FGFR)-1 and vascular endothelial growth factor receptor (VEGFR)-2; sulfatinib, an inhibitor of FGFR-1 and VEGFR-1, -2, -3; and SPP86, a RET-specific inhibitor. The effects of these compounds were evaluated in vitro in two human MTC cell lines (TT and MZ-CRC-1), and in vivo using xenografts of MTC cells in zebrafish embryos. SU5402, sulfatinib and SPP86 decreased cell viability. Sulfatinib and SPP86 significantly induced apoptosis in both cell lines. Sulfatinib and SPP86 inhibited the migration of TT and MZCRC-1 cells, while SU5402 was able to inhibit migration only in TT cells. In vivo we observed a significant reduction in TT cell-induced angiogenesis in zebrafish embryos after incubation with sulfatinib and SPP86. In conclusion, sulfatinib and SPP86 displayed a relevant antitumor activity both in vitro and in vivo. Moreover, this work suggests the potential utility of targeting FGFR and VEGFR signaling pathways as an alternative therapy for MTC.

Anti-angiogenic potential of bioactive phytochemicals from Helicteres isora targeting VEGFR-2 to fight cancer through molecular docking and molecular dynamics simulation

Cancer is one of the leading causes of death due to its very high rate of morbidity and mortality, and there is a constant demand of effective drugs for cancer therapy. Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a significant role as central modulator of angiogenesis and is targeted frequently for developing anti-angiogenic agents to fight cancer. Helicteres isora L. (Malvaceae) is reported to possess diverse medicinal properties including anti-cancer potentials. In the current investigation, 38 bioactive phytochemicals of H. isora were screened virtually to evaluate their anti-angiogenic potentials targeting VEGFR-2. The study unveiled three potential candidates such as, Diosgenin (-9.8 Kcal//mol), Trifolin (-8.4 Kcal/mol) and Yohimbine (-8.1 Kcal/mol) that showed favorable pharmacokinetic, pharmacodynamic and toxicity properties with no significant side effects. Molecular dynamics simulation employing 100 ns revealed noteworthy structural stability and compactness for all the top three candidates. The MM/GBSA binding free energy estimation corroborated the docking interactions where Yohimbine (-30.47 Kcal/mol) scored better than Diosgenin (-27.54 Kcal/mol) and Trifolin (-29.58 Kcal/mol). Target class prediction revealed enzymes in most of the cases and some FDA approved drugs were found as structurally similar analogs for Trifolin and Yohimbine. These findings could lead to the development of novel and effective anti-angiogenic agents. Communicated by Ramaswamy H. Sarma

Value of ultrasound-targeted vascular endothelial growth factor receptor-2 in non-invasive monitoring of anti-angiogenic response in nude mice with subcutaneous xenograft model

Objective: To investigate the value of contrast-enhanced ultrasound targeting vascular endothelial growth factor receptor-2 (VEGFR-2) in non-invasive monitoring of anti-angiogenesis response in subcutaneous transplantation tumor model of hepatocellular carcinoma in nude mice. Methods: Sixteen nude mice were randomly divided into control group and bevacizumab treatment group (treatment group). Two weeks later, the model of subcutaneous transplanted tumor was established. The mice in the treatment group were intratumorally injected with 0.2 mg bevacizumab, while the control group was given the same amount of saline, three times a week for 2 weeks. Ultrasound microbubbles targeting VEGFR-2 were prepared by biotin avidin method. Ultrasound examinations were performed before treatment, 7 days and 14 days after treatment, and the time intensity curve (TIC) was drawn to quantitatively analyze the differences of parameters with treatment time. The expression of CD31 in tumor tissues was detected by immunohistochemistry. Results: After 14 days of treatment, the volume of tumor tissue in the treatment group and the control group were (0.247±0.019) mm(3) and (0.307±0.031) mm(3,) respectively, the difference was statistically significant (P<0.01). After 7 days of treatment, the rise slope (K(1)), time to peak (TTP) and peak intensity (PI) of TIC curve in the treatment group were 3.77±0.62, (3.82±0.21) s and (24.35±3.34) dB, respectively, which were significantly different from 2.93±0.31, (4.47±0.50) s and (30.10±2.35) dB in the control group, respectively (P<0.05). Immunohistochemistry showed that the PI of contrast enhanced ultrasound was positively correlated with microvessel density (r(2)=0.898, P=0.017). Conclusions: The therapeutic effect of bevacizumab on tumor angiogenesis evaluated by contrast-enhanced ultrasound targeting VEGFR-2 is related to the differences of parameters such as K(1), TTP and PI of TIC index. Contrast enhanced ultrasound targeting VEGFR-2 is of great value in non-invasive monitoring of subcutaneous transplanted tumor model of hepatocellular carcinoma in nude mice.

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.

Discovery of novel indolyl-1,2,4-triazole hybrids as potent vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors with potential anti-renal cancer activity

Targeting VEGFR-2 signaling pathway is well-established as an important approach for the treatment of solid tumors, particularly renal cancer. Herein, novel indolyl-1,2,4-triazole hybrids were designed and synthesized as VEGFR-2 kinase inhibitors with potential anti-renal cancer activity. The structures of the newly synthesized compounds were confirmed based on their spectral and elemental analyses. The results of in vitro kinase assay indicated that all target compounds revealed submicromolar inhibition of VEGFR-2 kinase enzyme. Analogs 5c, 5d and 9b emerged as the most active compounds (IC50 = 0.034–0.064 µM), showing VEGFR-2 inhibitory activity much superior to that of sunitinib reference drug (IC50 = 0.075 µM). Moreover, compounds 5a, 8c, 9d, 12c were equipotent to sunitinib against VEGFR-2 kinase. Additionally, the most potent compounds were further examined for their anticancer activity against two human renal cancer cell lines. All screened compounds effectively inhibited the growth of the two tested cell lines with IC50 values spanning from sub-micromolar to low micromolar levels. Compounds 5b, 5d, 11c and 12c were three to five-fold more potent than sunitinib against CAKI-1 cell line. Analogue 8c was superior/comparable to sunitinib against CAKI-1/A498 cell lines. Moreover, compound 9d showed double potency of sunitinib against A498 cell line. Besides, compounds 8c and 12c demonstrated a safety profile much better than that of sunitinib against non-cancer human renal cells. As well, the docked models of title compounds revealed strong interactions with key residues within the active site of VEGFR-2 kinase.

Development of Human CBF1-Targeting Single-Stranded DNA Aptamers with Antiangiogenic Activity In Vitro.

C promoter binding factor 1 (CBF1) (alias RBPJ) is a critical transcription factor involved in Notch signaling. The activation of Notch signaling through CBF1 maintains the angiostatic state of endothelial cells suppressing angiogenesis, that is, the formation of new blood vessels. Vascular endothelial growth factor (VEGF) induces angiogenesis by promoting the proteasomal degradation of CBF1, in addition to endothelial cell proliferation. To date, angiogenic inhibitors targeting VEGF have been successfully used in clinics for cancer and age-related macular degeneration. Most antiangiogenic drugs, however, only target VEGF or VEGF receptors. In this study, to expand the repertoire of antiangiogenic therapeutics, we developed 15 single-stranded deoxyribonucleic acid (ssDNA) aptamers capable of binding to CBF1 with high affinity (Kd; 10-300 nM). To this end, systematic evolution of ligands by the exponential enrichment (SELEX) method was applied. One of the CBF1-binding ssDNA aptamers, Apt-3, inhibited angiogenesis through the activation of Notch signaling in vitro. We found that Apt-3 directly interacted with the LAG1 domain of CBF1. We suggest that the Apt-3 ssDNA aptamer may contribute to the development of a novel angiogenic inhibitor, which does not target VEGF.

VEGF receptor targeted imaging of angiogenic response to limb ischemia in diabetic vs. non-diabetic Yucatan minipigs

BackgroundNew therapies to treat diabetic peripheral artery disease (PAD) require target-specific non-invasive imaging modalities to follow efficacy. As a translational study, we performed targeted imaging of receptors for vascular endothelial growth factor (VEGF) in response to anterior femoral artery occlusion (FAO) in Yucatan minipigs and compare the normal response to response in diabetic Yucatan minipigs.MethodsEleven Yucatan minipigs, 6 non-diabetic (non-D) and 5 purpose bred diabetic (D) (Sinclair, Auxvasse MO), underwent intravascular total occlusion of the anterior femoral artery (FA). At days 1 and 28, pigs underwent SPECT/CT 201Tl hindlimb perfusion imaging and at day 7 were injected with [99mTc]DOTA-PEG-scVEGF (scV/Tc) tracer targeting VEGF receptor, and underwent biopsies of the hindlimb muscles for gamma counting and histology, followed by imaging. One day after the final scan, pigs underwent contrast angiography of the lower extremities. Counts from scans were converted to percentage injected activity (%IA).ResultsPerfusion was lower in the occluded hindlimb compared to non-occluded on day 1 in both the D and non-D pigs. At day 7, scV/Tc count ratio of counts from ROIs drawn in proximal gastrocnemius muscle for the occluded over non-occluded limb was significantly higher in non-D vs. D pigs (1.32 ± 0.06 vs. 1.04 ± 0.13, P = 0.02) reflecting higher level of angiogenesis. Perfusion increased between days 1 and 28 in the muscles in the occluded limb for the non-diabetic pigs while the diabetic pig showed no increase (+ 0.13 ± 0.08 %IA vs. − 0.13 ± 0.11, P = 0.003). The anterior FA showed poor contrast filling beyond occluder and qualitatively fewer bridging collaterals compared to non-D pigs at 28 days.ConclusionVEGF receptor targeted imaging showed the effects of diabetes to suppress angiogenesis in response to occlusion of the anterior femoral artery of purpose bred diabetic Yucatan minipigs and indicates potential applicability as a marker to follow efficacy of novel therapies to improve blood flow by stimulating angiogenesis in diabetic PAD.

A self-assembling peptide targeting VEGF receptors to inhibit angiogenesis

ABSTRACT Vascular endothelial growth factor (VEGF)-vascular endothelial growth factor receptor (VEGFR) pathways are essential in tumor angiogenesis, growth and metastasis. Studies on anti-angiogenic therapy have been mostly focused on the blockage of VEGF-VEGFR pathways. We report an extracellularly transformable peptide-based nanomaterial to develop artificial extracellular matrix (ECM)-like networks for high-efficient blockage of natural VEGF-VEGFR interactions. The transformable peptide-based nanomaterial transforms from nanoparticles into nanofibers upon binding to VEGFR in solution. In addition, the transformable peptide-based nanomaterial forms ECM-like fibrous networks on VEGFR overexpressed cells, inhibiting the VEGF-VEGFR interactions and the subsequent angiogenesis. The tube formation is reduced by nearly 85.1% after treatment. This strategy shows excellent potential for anti-angiogenesis, and inhibition of tumor invasion and metastasis.

Molecular docking and interactions between vascular endothelial growth factor (VEGF) receptors and phytochemicals: An in-silico study

Abstract Vascular endothelial growth factor (VEGF) is a mitogen, which plays a vital role in angiogenesis and neovascularization. Numerous medications have been developed to inhibit VEGF in cancer, because of its significance in tumor development and its survival. Hence, the current study is focused on identifying phytocompounds to directly target VEGF receptors using in-silico approach. In this study, a computational approach has been applied to elucidate the role of the phytocompounds in inhibition of angiogenesis through modulation of VEGF receptors responsible for angiogenesis. Molecular docking analysis was performed using AutoDock 4.2 software. In addition, the in silico ADMET analysis was carried out to predict the pharmacokinetic properties. Among the 50 phytocompounds tested, 6 compounds (Eriodyctiol, Epicatechin, Scutellarin, Ginkgolide A, Tetrandrine and Bassic acid) showed a better interaction profile than standard drug (Triamcinolone). Our study suggests a potential therapeutic effect of these phytocompounds for inhibiting angiogenesis, thereby providing inkling for novel drug design for proangiogenic diseases such as DR.

Targeting Fluorescent Nanodiamonds to Vascular Endothelial Growth Factor Receptors in Tumor.

The increased expression of vascular endothelial growth factor (VEGF) and its receptors is associated with angiogenesis in a growing tumor, presenting potential targets for tumor-selective imaging by way of targeted tracers. Though fluorescent tracers are used for targeted in vivo imaging, the lack of photostability and biocompatibility of many current fluorophores hinder their use in several applications involving long-term, continuous imaging. To address these problems, fluorescent nanodiamonds (FNDs), which exhibit infinite photostability and excellent biocompatibility, were explored as fluorophores in tracers for targeting VEGF receptors in growing tumors. To explore FND utility for imaging tumor VEGF receptors, we used click-chemistry to conjugate multiple copies of an engineered single-chain version of VEGF site-specifically derivatized with trans-cyclooctene (scVEGF-TCO) to 140 nm FND. The resulting targeting conjugates, FND-scVEGF, were then tested for functional activity of the scVEGF moieties through biochemical and tissue culture experiments and for selective tumor uptake in Balb/c mice with induced 4T1 carcinoma. We found that FND-scVEGF conjugates retain high affinity to VEGF receptors in cell culture experiments and observed preferential accumulation of FND-scVEGF in tumors relative to untargeted FND. Microspectroscopy provided unambiguous determination of FND within tissue by way of the unique spectral shape of nitrogen-vacancy induced fluorescence. These results validate and invite the use of targeted FND for diagnostic imaging and encourage further optimization of FND for fluorescence brightness.

Tc-99m and Fluorescence-Labeled Anti-Flt1 Peptide as a Multimodal Tumor Imaging Agent Targeting Vascular Endothelial Growth Factor-Receptor 1.

We developed a Tc-99m and fluorescence-labeled peptide, Tc-99m TAMRA-GHEG-ECG-GNQWFI, to target tumor cells, and evaluated the diagnostic performance as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-GNQWFI was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-GNQWFI with Tc-99m was done using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with U87MG tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-GNQWFI complexes were prepared in high yield (> 95%). The K d of Tc-99m TAMRA-GHEG-ECG-GNQWFI determined by saturation binding was 29.5 ± 4.5nM. Confocal microscopy images of U87MG cells incubated with TAMRA-GHEG-ECG-GNQWFI showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-GNQWFI in tumors. Tumor uptake was effectively blocked by the co-injection of an excess concentration of GNQWFI. Specific uptake of Tc-99m TAMRA-GHEG-ECG-GNQWFI was assessed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In vivo and in vitro studies revealed substantial and specific uptake of Tc-99m TAMRA-GHEG-ECG-GNQWFI in tumor cells. Tc-99m TAMRA-GHEG-ECG-GNQWFI could be a good candidate dual-modality imaging agent for tumors.

Abstract 2047: N-glycosylation modulates endothelial cell receptor tyrosine kinase VEGFR-2 ligand-dependent activation and signaling

Abstract Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial receptor tyrosine kinase (RTK), is essential for tumor angiogenesis in tumors of diverse origin. The extracellular domain of VEGFR-2 contains seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites. Changes in glycosylation influenced by hypoxia, the shunting of glycolytic intermediates via the hexosamine biosynthesis pathway into glycan synthesis, and pro-inflammatory cytokines have the potential to alter VEGFR-2 signaling in tumors. However, the role of glycosylation in VEGFR-2 signaling and function remain largely unknown. The objective of this study is to determine the functional importance of N-glycosylation in VEGFR-2 activation and angiogenic signaling. Porcine aortic endothelial (PAE) cells with ectopic expression of either wild type VEGFR-2, or one in a series of VEGFR-2 constructs with single glycosylation site mutations in the ligand binding domain, were treated with VEGF-A for 0, 5, 10 or 30 minutes. VEGFR-2 tyrosine phosphorylation was measured via Western blot with an anti-pTyr-1054-VEGFR-2 antibody to assess activation, and with anti-pTyr-1175-VEGFR-2 and anti-pTyr-1212-VEGFR-2 antibodies. Activation of key molecules involved in downstream signaling of VEGFR-2 was also monitored. To establish the glycosylation status of VEGFR-2 available for ligand-mediated signaling, we isolated plasma membrane localized VEGFR-2, performed enzymatic proteolysis, enriched glycopeptides via hydrophilic interaction liquid chromatography (HILIC), and analyzed glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation. Mutation of one N-glycosylation site near the VEGF binding site increased ligand-dependent phosphorylation (at Tyr-1054, Tyr-1175 and Tyr-1212) within 5 min of ligand addition, and initial observations suggest that this resulted in higher levels of activated c-Src compared to wild type VEGFR-2, which may have implications for endothelial cell proliferation and migration. However, phosphorylation of other downstream signaling targets including Akt and PLCγ1 was not affected. Mutations at other N-glycosylation sites near the VEGF ligand binding site did not appear to impact ligand-mediated activation of VEGFR-2, suggesting that glycans located at certain N-linked sites may influence ligand-mediated activation while others have minimal impact. Targeting VEGFR-2 N-glycosylation may be a valuable strategy to inhibit tumor angiogenesis. This research is supported by NIH grants P41 GM104603, R01 CA191970, and F32 CA196157. Novel aspect: These results demonstrate that site-specific N-glycosylation of VEGFR-2 modulates ligand-mediated phosphorylation of key VEGFR-2 tyrosine residues involved in downstream signaling. Citation Format: Kevin B. Chandler, Deborah R. Leon, Jenevieve Kuang, Rosana D. Meyer, Nader Rahimi, Catherine E. Costello. N-glycosylation modulates endothelial cell receptor tyrosine kinase VEGFR-2 ligand-dependent activation and signaling [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 2047.

PBA2 exhibits potent anti-tumor activity via suppression of VEGFR2 mediated-cell proliferation and angiogenesis

VEGFR2 (vascular endothelial growth factor receptor 2) is the main trigger of VEGF-mediated angiogenic signal and targeting VEGFR2 pathway to inhibit tumor angiogenesis represents a promising strategy for cancer therapy. We elucidated that a novel compound, PBA2 exhibited potent anti-tumor effects both in vitro and in vivo with limited toxicity. ELISA assay revealed that PBA2 had a strong suppressive activity against VEGFR2 related to angiogenesis. Furthermore, PBA2 considerably disrupted tube formation of endothelial cells in vitro and systemic administration of PBA2 exerted decreased tumor angiogenesis in vivo. Functional tests demonstrated that PBA2 concentration-dependently impeded the migration and proliferation of endothelial cells. PBA2 had no effects on the expression level of VEGF both in the detected cancer cells and endothelial cells. VEGFR2 and its downstream Akt and Erk pathways were blocked by PBA2 in a concentration-dependent manner both in vitro and in vivo. Overall, we first demonstrated that PBA2, targeting VEGFR2 related to angiogenesis, presented remarkable anti-angiogenic and anti-tumor activities through attenuating VEGFR2 mediated pathway in vitro and in vivo with limited toxicity. These observations posed that PBA2 could be a potential drug candidate for developing anti-angiogenic tyrosine kinase inhibitor in cancer therapy.