Interactions of Nanoscale Self-Assembled Peptide-Based Assemblies with Glioblastoma Cell Models and Spheroids.

Peptide nanoassemblies have garnered remarkable importance in the development of novel nanoscale biomaterials for drug delivery into tumor cells. Taking advantage of receptor mediated recognition of two known peptides, angiopep-2 (TFFYGGSRGKRNNFKTEEY) and A-COOP-K (ACGLSGLC10 VAK) that bind to the over-expressed receptors low density lipoprotein (LRP-1) and fatty acid binding protein (FABP3) respectively, we have developed new peptide conjugates by combining the anti-inflammatory, antitumor compound azelaic acid with angiopep-2, which efficiently self-assembled into nanofibers. Those nanofibers were then functionalized with the A-COOP-K sequence and formed supramolecular hierarchical structures that were found to entrap the chemotherapeutic drug doxorubicin efficaciously. Furthermore, the nanoassemblies were found to release the drug in a dose-dependent manner and showed a stepwise increase over a period of 2 weeks under acidic conditions. Two cell lines (U-87-MG and U-138-MG) were utilized as models for glioblastoma cells grown in the presence of serum and under serum-free conditions to mimic the growth conditions of natural tumors. The drug entrapped assemblies were found to inhibit the cell proliferation of both U-87 and U-138MG glioblastoma cells. Three dimensional spheroids of different sizes were grown to mimic the tumors and evaluate the efficacy of drug release and internalization. Our results indicated that the nanoassemblies were found to have higher internalization of DOX and were well-spread throughout the spheroids grown, particularly under serum-free conditions. The nanoassemblies also displayed blood-brain barrier penetration when tested with a multicellular in vitro model. Such self-assembled nanostructures with targeting ability may provide a suitable platform for the development of new peptide-based biomaterials that can provide more insights about the mechanistic approach for drug delivery for not only 2D cell cultures but also 3D tumoroids that mimic the tumor microenvironments.

An investigation of binding interactions of tumor-targeted peptide conjugated polyphenols with the kinase domain of ephrin B4 and B2 receptors.

Recent studies have shown that Ephrin receptors may be upregulated in several types of cancers including breast, ovarian and endometrial cancers, making them a target for drug design. In this work, we have utilized a target-hopping approach to design new natural product-peptide conjugates and examined their interactions with the kinase-binding domain of EphB4 and EphB2 receptors. The peptide sequences were generated through point mutations of the known EphB4 antagonist peptide TNYLFSPNGPIA. Their anticancer properties and secondary structures were analyzed computationally. Conjugates of most optimum of peptides were then designed by binding the N-terminal of the peptides with the free carboxyl group of the polyphenols sinapate, gallate and coumarate, which are known for their inherent anticancer properties. To investigate if these conjugates have a potential to bind to the kinase domain, we carried out docking studies and MMGBSA free energy calculations of the trajectories based on the molecular dynamics simulations, with both the apo and the ATP bound kinase domains of both receptors. In most cases binding interactions occurred within the catalytic loop region, while in some cases the conjugates were found to spread out across the N-lobe and the DFG motif region. The conjugates were further tested for prediction of pharmacokinetic properties using ADME studies. Our results indicated that the conjugates were lipophilic and MDCK permeable with no CYP interactions. These findings provide an insight into the molecular interactions of these peptides and conjugates with the kinase domain of the EphB4 and EphB2 receptor. As a proof of concept, we synthesized and carried out SPR analysis with two of the conjugates (gallate-TNYLFSPNGPIA and sinapate-TNYLFSPNGPIA). Results indicated that the conjugates showed higher binding with the EphB4 receptor and minimal binding to EphB2 receptor. Sinapate-TNYLFSPNGPIA showed inhibitory activity against EphB4. These studies reveal that some of the conjugates may be developed for further investigation into in vitro and in vivo studies and potential development as therapeutics.

Molecular dynamics, MMGBSA, and docking studies of natural products conjugated to tumor-targeted peptide for targeting BRAF V600E and MERTK receptors.

Recent studies have revealed that MERTK and BRAF V600E receptors have been found to be over-expressed in several types of cancers including melanoma, making these receptors targets for drug design. In this study, we have designed novel peptide conjugates with the natural products vanillic acid, thiazole-2-carboxylic acid, cinnamic acid, theanine, and protocatechuic acid. Each of these compounds was conjugated with the tumor targeting peptide sequence TAASGVRSMH, known to bind to NG2 and target tumor neovasculature. We examined their binding affinities and stability with MERTK and BRAF V600E receptors using molecular docking and molecular dynamics studies. Compared to the neat compounds, the peptide conjugates displayed higher binding affinity toward both receptors. In the case of MERTK, the most stable complexes were formed with di-theaninate-peptide, vanillate-peptide, and thiazole-2-amido peptide conjugates and binding occurred in the hinge region. Additionally, it was discovered that the peptide alone also had high binding ability and stability with the MERTK receptor. In the case of BRAF V600E, the peptide conjugates of protocatechuate, vanillate and thiazole-2-amido peptide conjugates showed the formation of the most stable complexes and binding occurred in the ATP binding cleft. Further analysis revealed that the number of hydrogen bonds and hydrophobic interactions played a critical role in enhanced stability of the complexes. Docking studies also revealed that binding affinities for NG2 were similar to MERTK and higher for BRAF V600E. MMGBSA studies of the trajectories revealed that the protocatechuate-peptide conjugate showed the highest binding energy with BRAF V600E while the peptide-TAASGVRSMH showed the highest binding energy with MERTK. ADME studies revealed that each of the compounds showed medium to high permeability toward MDCK cells and were not hERG blockers. Furthermore, the conjugates were not CYP inhibitors or substrates, but they were found to be Pgp substrates. Our results indicated that the protocatechuate-TAASGVRSMH, thiazole-2-amido-TAASGVRSMH, and vanillate-TAASGVRSMH conjugates may be furthered developed for in vitro and in vivo studies as novel tumor targeting compounds for tumor cells over-expressing BRAF V600E, while di-theaninate-amido-TAASGVRSMH and thiazole-2-amido-TAASGVRSMH conjugates may be developed for targeting MERTK receptors. These studies provide insight into the molecular interactions of natural product-peptide conjugates and their potential for binding to and targeting MERTK and BRAF V600E receptors in developing new therapeutics for targeting cancer.