Computational Design of TrkB Peptide Inhibitors and Their Biological Effects on Ovarian Cancer Cell Lines

Abstract There are large numbers of different intracellular signaling pathways regulated by Tyrosine kinases (Trk) receptors. Trk receptors, especially TrkB, are also frequently overexpressed in a variety of human malignant tumors. In this study, we have computationally designed small peptide-based inhibitors of TrkB and investigated their effects on the proliferation and apoptosis of two ovarian cancer cell lines. Molecular docking of TrkB with its ligand and antagonist, BDNF and Cyclotraxin B respectively, was carried out using HADDOCK program. A peptide library was constructed based on the critical residues involved in the TrkB binding site. After docking and optimization, two selected peptides were purchased and their effects on the viability and apoptosis of the cells were evaluated by performing MTT (3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) test and flow cytometry assay. Subsequently, the levels of expression and phosphorylation statues of TrkB and its two downstream genes including MAPK3 and eIF4E were assessed with western blot. We found that designed peptides effectively reduced TrkB, MAPK3 and eIF4E phosphorylation, reduced cell viability and induced apoptosis in the treated cells when compared to untreated cells. In conclusion, the BDNF/TrkB signaling is shown to be attenuated substantially in the presence of peptide inhibitors suggesting a strong inhibitory potential of the designed peptides for Trk famil

TCR-like CARs and TCR-CARs targeting neoepitopes: an emerging potential

Neoepitopes or neoantigens are a spectrum of unique mutations presented in a particular patient's tumor. Neoepitope-based adoptive therapies have the potential of tumor eradication without undue damaging effect on normal tissues. In this context, methods based on the T cell receptor (TCR) engineering or chimeric antigen receptors (CARs) have shown great promise. This review focuses on the TCR-like CARs and TCR-CARs directed against tumor-derived epitopes, with a concerted view on neoepitopes. We also address the current limitations of the field to know how to harness the full benefits of this approach and thereby design a sustained and specific antitumor therapy

Update on immune‐based therapy strategies targeting cancer stem cells

Abstract Accumulating data reveals that tumors possess a specialized subset of cancer cells named cancer stem cells (CSCs), responsible for metastasis and recurrence of malignancies, with various properties such as self‐renewal, heterogenicity, and capacity for drug resistance. Some signaling pathways or processes like Notch, epithelial to mesenchymal transition (EMT), Hedgehog (Hh), and Wnt, as well as CSCs' surface markers such as CD44, CD123, CD133, and epithelial cell adhesion molecule (EpCAM) have pivotal roles in acquiring CSCs properties. Therefore, targeting CSC‐related signaling pathways and surface markers might effectively eradicate tumors and pave the way for cancer survival. Since current treatments such as chemotherapy and radiation therapy cannot eradicate all of the CSCs and tumor relapse may happen following temporary recovery, improving novel and more efficient therapeutic options to combine with current treatments is required. Immunotherapy strategies are the new therapeutic modalities with promising results in targeting CSCs. Here, we review the targeting of CSCs by immunotherapy strategies such as dendritic cell (DC) vaccines, chimeric antigen receptors (CAR)‐engineered immune cells, natural killer‐cell (NK‐cell) therapy, monoclonal antibodies (mAbs), checkpoint inhibitors, and the use of oncolytic viruses (OVs) in pre‐clinical and clinical studies. This review will mainly focus on blood malignancies but also describe solid cancers

Rational design of DKK3 structure-based small peptides as antagonists of Wnt signaling pathway and in silico evaluation of their efficiency.

Dysregulated Wnt signaling pathway is highly associated with the pathogenesis of several human cancers. Dickkopf proteins (DKKs) are thought to inhibit Wnt signaling pathway through binding to lipoprotein receptor-related protein (LRP) 5/6. In this study, based on the 3-dimensional (3D) structure of DKK3 Cys-rich domain 2 (CRD2), we have designed and developed several peptide inhibitors of Wnt signaling pathway. Modeller 9.15 package was used to predict 3D structure of CRD2 based on the Homology modeling (HM) protocol. After refinement and minimization with GalaxyRefine and NOMAD-REF servers, the quality of selected models was evaluated utilizing VADAR, SAVES and ProSA servers. Molecular docking studies as well as literature-based information revealed two distinct boxes located at CRD2 which are actively involved in the DKK3-LRP5/6 interaction. A peptide library was constructed conducting the backrub sequence tolerance scanning protocol in Rosetta3.5 according to the DKK3-LRP5/6 binding sites. Seven tolerated peptides were chosen and their binding affinity and stability were improved by some logical amino acid substitutions. Molecular dynamics (MD) simulations of peptide-LRP5/6 complexes were carried out using GROMACS package. After evaluation of binding free energies, stability, electrostatic potential and some physicochemical properties utilizing computational approaches, three peptides (PEP-I1, PEP-I3 and PEP-II2) demonstrated desirable features. However, all seven improved peptides could sufficiently block the Wnt-binding site of LRP6 in silico. In conclusion, we have designed and improved several small peptides based on the LRP6-binding site of CRD2 of DKK3. These peptides are highly capable of binding to LRP6 in silico, and may prevent the formation of active Wnt-LRP6-Fz complex

Multi-targeting of K-Ras domains and mutations by peptide and small molecule inhibitors.

K-Ras activating mutations are significantly associated with tumor progression and aggressive metastatic behavior in various human cancers including pancreatic cancer. So far, despite a large number of concerted efforts, targeting of mutant-type K-Ras has not been successful. In this regard, we aimed to target this oncogene by a combinational approach consisting of small peptide and small molecule inhibitors. Based on a comprehensive analysis of structural and physicochemical properties of predominantly K-Ras mutants, an anti-cancer peptide library and a small molecule library were screened to simultaneously target oncogenic mutations and functional domains of mutant-type K-Ras located in the P-loop, switch I, and switch II regions. The selected peptide and small molecule showed notable binding affinities to their corresponding binding sites, and hindered the growth of tumor cells carrying K-RasG12D and K-RasG12C mutations. Of note, the expression of K-Ras downstream genes (i.e., CTNNB1, CCND1) was diminished in the treated Kras-positive cells. In conclusion, our combinational platform signifies a new potential for blockade of oncogenic K-Ras and thereby prevention of tumor progression and metastasis. However, further validations are still required regarding the in vitro and in vivo efficacy and safety of this approach

A schematic representation of BoxI (Left) and BoxII (Right) peptide optimization.

<p>The initial peptides (I) were tolerated (T) using Backrub and sequence tolerance protocols conducted by Rosetta package. Each position of peptide was substituted with other residues (substitutions with BLOSUM62 score -4 were omitted to avoid deleterious substitutions). The blue, orange and yellow colors indicate unfavorable (Δ<i>G</i>_{<i>interaction</i>} of resulted peptide > Δ<i>G</i>_{<i>interaction</i>} of input peptide), favorable (Δ<i>G</i>_{<i>interaction</i>} of resulted peptide < Δ<i>G</i>_{<i>interaction</i>} of input peptide) and neutral substitutions (Δ<i>G</i>_{<i>interaction</i>} of resulted peptide ≊ Δ<i>G</i>_{<i>interaction</i>} of input peptide), respectively, and the green color represents non-mutated residues. Substitutions that caused a <100 change in the value of interaction weighted score (calculated by ClusPro) were considered as neutral. All possible combinations of favorable substitutions were generated and the best peptides were selected among them.</p

Evaluation of structural fluctuations and surface accessibility.

<p>(<b>A</b>) The RMSF of each peptide-<i>LRP6</i> complex as a function of the residue number in the <i>LRP6</i> protein. (<b>B</b>) The SASA values for Wnt-binding residues of <i>LRP6</i> with or without each designed peptides.</p

Residues of <i>LRP6</i> interacting with the peptides as predicted by LIGPLOT.

<p>Bold residues: Wnt- and DKK-binding, bold and underline: DKK-binding.</p

Validation of CRD2 model by several methods.

<p>(<b>A</b>) Ramachandran plot. The most favored, additionally allowed, generously allowed and disallowed regions are shown in red, yellow, beige and white colors, respectively. (<b>B</b>) Structural alignment of <i>DKK3C</i> (gray) and 2JTK pdb (blue). (<b>C</b>) ProSA Z-score plot of modeled 3D structure of <i>DKK3C</i>. The position of this model among experimentally solved protein structures is shown in an open red circle. (<b>D</b>) Local model quality by plotting energies as a function of amino acid sequence position. Generally, positive values correspond to problematic parts of the input structure. (<b>E</b>) Sequence and secondary structure alignment of <i>DKK3C</i> and mouse dkk2 (PDB ID: 2JTK) conducted by ESPript 3.0 (<a href="http://espript.ibcp.fr/ESPript/ESPript/" target="_blank">http://espript.ibcp.fr/ESPript/ESPript/</a>).</p