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Identification and characterization of agnuside, a natural proangiogenic small molecule.

ABSTRACT: Due to its important role in regulating angiogenesis, vascular homeostasis and remodeling, and arteriogenesis in blood vascular and lymphatic endothelial cells, VEGFR2 stimulation has demonstrated promise in preclinical studies as an endovascular treatment for ischemic myocardial and peripheral disease. However, the short half-life of protein- and cytokine-based strategies and transduction inefficiency of vector-based modalities have hindered its clinical therapeutic applications. In the present study, we used a streamlined bioinformatics strategy combining ligand-based pharmacophore development and validation, virtual screening, and molecular docking to identify agnuside, a non-toxic, natural small molecule extract of Vitex agnus-castus possessing strong binding affinity, druggable physiochemical properties, and conformationally stable hydrogen bond and hydrophobic interactions with catalytically important residues within VEGFR2's active and allosteric sites. In-vitro proliferation, tube formation, and scratch wound migration assays provide evidence that agnuside promotes endothelial cell angiogenesis. Agnuside increases HUVEC proliferation with an EC50 of 1.376??g/mL, stimulates tubulogenesis dose-dependently, and increases scratch wound migration rate. An additional angiogenesis assay suggests that agnuside may actively compete with a VEGFR2 inhibitor for VEGFR2 binding site occupancy to increase total length and branching length of HUVEC tubular networks. Chemometric analysis of molecular interaction fields (MIFs) by partial least squares (PLS)-derived quantitative structure activity relationship (QSAR) analysis and MIF contours provides the framework for the formulation of agnuside analogues possessing greater potency. Our research supports that agnuside may be a lead molecule for therapeutic angiogenesis.

SUBMITTER: Pillarisetti P

PROVIDER: S-EPMC6287603 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Identification and characterization of agnuside, a natural proangiogenic small molecule.

Pillarisetti Piyush P Myers Kenneth A KA

European journal of medicinal chemistry 20181005

Due to its important role in regulating angiogenesis, vascular homeostasis and remodeling, and arteriogenesis in blood vascular and lymphatic endothelial cells, VEGFR2 stimulation has demonstrated promise in preclinical studies as an endovascular treatment for ischemic myocardial and peripheral disease. However, the short half-life of protein- and cytokine-based strategies and transduction inefficiency of vector-based modalities have hindered its clinical therapeutic applications. In the present ...[more]

PMID: 30340142

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Project description:Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV reporter strain. A large-scale screen and subsequent direct and orthogonal counterscreens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry.IMPORTANCE Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could address this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV entry inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.

Dataset Information

Identification and characterization of agnuside, a natural proangiogenic small molecule.

Publications

Identification and characterization of agnuside, a natural proangiogenic small molecule.

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