Point Mutation Effect on Diphtheria Toxin- based Immunotoxin against Granulocyte-Colony Stimulating Factor Receptor In silico as Targeted Therapeutic Strategy
Point Mutation Effect on Diphtheria Toxin- based Immunotoxin against Granulocyte-Colony Stimulating Factor Receptor In silico as Targeted Therapeutic Strategy
Ghazale Faridfar,1Mehdi Zeinoddini,2,*Shadi Jabbarzadeh,3
1. Department of Bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran 2. Department of Bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran 3. Department of veterinary medicine ,Azad Islamic University of Karaj, Karaj, Iran
Introduction: Recent advances in the design and administration of immunotoxins are currently in clinical development. Immunotoxin drugs delivery is a non-invasive treatment that inserts from specific receptors on the surface of cancer cells and eliminates them purposefully. These recombinant proteins carry a toxin fused to a targeting cell. Following the ligand-receptor binding of the target cells, the toxin invades the cytoplasm and destroys them. An important feature of the recombinant protein is its ability to isolate ligands from the predictable catalytic domain. Hence, bacteriotherapy development knowledge of cancer has been regarded as a novel and effective technique. Accordingly, Diphtheria Toxin (DT) is broadly applied due to its easy expression, high activity, and minimal side effects for therapeutic. In this initial analysis, we have provided insight into the structural and functional properties of fusion DT387GCSF protein and its receptor. Accordingly, DT387GCSF immunotoxin was contained two portions: truncated diphtheria toxin (DT) and granulocyte-colony stimulating factor (G-CSF). In this study, we first focus on the structure and function of the fusion protein of diphtheria immunotoxin, then we will prepare for further processes. Our study aimed to develop recombinant DT387GCSF protein and evaluate its structural and functional characteristics relative to its receptor.
Methods: According to the scheme objective, the DT387GCSF sequence and its receptor were downloaded in the FASTA format. Following, the three-dimensional structure of the protein was predicted through the DE NOVO MODELING method using the ITASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). Next, refinement of the predicted models was obtained for efficient structure using 3D refine server. To validate the presented models, the files obtained were evaluated using the server http://molprobity.biochem.duke.edu/. It should be noted the energy level of all PDB files was diminished to the most stable situation using SPDBV_4.10_PC software. The proteins geometry quality was also evaluated based on the Ramachandran plot using the RAMPAGE server (https://servicesn.mbi.ucla.edu/SAVES/). The structure modeling was created point mutations (C387S) using Molegro Virtual Docker software. Finally, protein-protein interaction was investigated using the Zdock server (http://zdock.umassmed.edu/ ) and HEX software. Ultimately, the tendency to connect the mutant with the native ligand was compared by the available servers.
Results: The Comparison of binding energy in the native and Ser-387 Mutant samples with their receptor demonstrated no significant difference in the binding affinity of the ligand at their receptor. The outcome also indicated that point mutation improved the accurate functional structure. Generally, the software analysis also explained there was no meaningful distinction in an efficient connection of the mutant protein and its receptor compared to the prime model.
Conclusion: Unlike sulfhydryl (SH-groups) side chains of cysteine, the results demonstrated that the point mutation (ser-387 mutant) doesn't impair the receptor-ligand interaction. Moreover, this process also increases drug production and efficiency. We concluded that DT387GCSF might be useful as a targeted toxin in G-CSF responsive cells. Besides, this review may provide an advantage to gene-based DT therapeutics and novel therapeutic applications in prospect.