Table1_Fiber manipulation and post-assembly nanobody conjugation for adenoviral vector retargeting through SpyTag-SpyCatcher protein ligation.docx

For adenoviruses (Ads) to be optimally effective in cancer theranostics, they need to be retargeted toward target cells and lose their natural tropism. Typically, this is accomplished by either engineering fiber proteins and/or employing bispecific adapters, capable of bonding Ad fibers and tumor antigen receptors. This study aimed to present a simple and versatile method for generating Ad-based bionanoparticles specific to target cells, using the SpyTag-SpyCatcher system. The SpyTag peptide was inserted into the HI loop of fiber-knob protein, which could act as a covalent anchoring site for a targeting moiety fused to a truncated SpyCatcher (SpyCatcherΔ) pair. After confirming the presence and functionality of SpyTag on the Ad type-5 (Ad5) fiber knob, an adapter molecule, comprising of SpyCatcherΔ fused to an anti-vascular endothelial growth factor receptor 2 (VEGFR2) nanobody, was recombinantly expressed in Escherichia coli and purified before conjugation to fiber-modified Ad5 (fmAd5). After evaluating fmAd5 detargeting from its primary coxsackie and adenovirus receptor (CAR), the nanobody-decorated fmAd5 could be efficiently retargeted to VEGFR2-expressing 293/KDR and human umbilical vein endothelial (HUVEC) cell lines. In conclusion, a plug-and-play platform was described in this study for detargeting and retargeting Ad5 through the SpyTag-SpyCatcher system, which could be potentially applied to generate tailored bionanoparticles for a broad range of specific targets; therefore, it can be introduced as a promising approach in cancer nanotheranostics.</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

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

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

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

Binding model and surface potentials of <i>DKK1</i>/3 and <i>LRP6</i>.

<p>(<b>A</b>) <i>DKK3C</i>-<i>LRP6</i> and (<b>B</b>) <i>DKK1</i>-<i>LRP6</i> complexes. <i>DKK1</i>-<i>LRP6</i> complex was obtained from PDB (ID: 3S2K), while <i>DKK3C</i>-<i>LRP6</i> is the result of ClusPro docking server. The surface potential of binding sites (open circles) calculated by PyMOL software are shown in the bottom of figure. Blue, red and gray colors infer the positive, negative and hydrophobic regions, respectively.</p

Calculation of electrostatic potentials around the peptides and H-bond numbers of peptide-<i>LRP6</i> complexes.

<p>(<b>A</b>) Electrostatic potentials around the peptides upon binding to <i>LRP6</i>. Hydrophobic, positive and negative potentials are shown in gray, blue and red colors, respectively. The highest level of blue and red colors is observed in regions with electrostatic potentials ≥2 and ≤-2, respectively. (<b>B</b>) The formation of H-bonds between the peptides and <i>LRP6</i> during the production phases of MD simulation.</p

3D structures of whole <i>DKK3</i> protein along with CRD2 of <i>DKK1</i> and <i>DKK3</i>.

<p>(<b>A</b>) Cartoon representation of 3D structure of <i>DKK3</i>; N-terminal, CRD2 and C-terminal regions are shown in green, orange and blue colors, respectively. (<b>B</b>) The <i>LRP6</i>-binding residues (blue) of CRD2 in <i>DKK1</i>. (<b>C</b>) The <i>LRP6</i> binding residues of <i>DKK3</i>. A 6-mer box (red) of positively charged residues in CRD2 of <i>DKK1</i> has an important role in <i>DKK1</i>-<i>LRP6</i> interactions, while this site in CRD2 of <i>DKK3</i> (red) has considerably changed with several non-conservative substitutions and has no role in <i>DKK3C</i>-<i>LRP6</i> interactions.</p

Evaluation of peptide-<i>LRP6</i> free energies, contacts and interacting surface.

<p>Evaluation of peptide-<i>LRP6</i> free energies, contacts and interacting surface.</p

RMSD and Rg plots of peptide-<i>LRP6</i> complexes after 20 ns MD simulations.

<p>(<b>A</b>) RMSD of the backbone atoms and (<b>B</b>) Rg plots of PEPI1-<i>LRP6</i> (green), PEPI2-<i>LRP6</i> (red), PEPI3-<i>LRP6</i> (magenta), PEPI4-<i>LRP6</i> (orange), PEPII1-<i>LRP6</i> (brown), PEPII2-<i>LRP6</i> (blue) and PEPII3-<i>LRP6</i> (cyan) complexes as a function of time.</p