TITLE: Computational design of novel nanobodies targeting the receptor binding domain of variants of concern of SARS-CoV-2
AUTHORS: Phoomintara Longsomboon, Thanyada Rungrotmongkol, Nongluk Plongthongkum, Kittikhun Wangkanont, Peter Wolschann, Rungtiva P. Poo-arporn

[DESCRIPTION]


The COVID-19 pandemic has created an urgent need for effective therapeutic
and diagnostic strategies to manage the disease caused by the severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the emergence of
numerous variants of concern (VOCs) has made it challenging to develop targeted
therapies that are broadly specific in neutralizing the virus. In this study, we
aimed to develop neutralizing nanobodies (Nbs) using computational techniques
that can effectively neutralize the receptor-binding domain (RBD) of SARS-CoV-2
VOCs. We evaluated the performance of different protein-protein docking
programs and identified HDOCK as the most suitable program for Nb/RBD docking
with high accuracy. Using this approach, we designed 14 novel Nbs with high
binding affinity to the VOC RBDs. The Nbs were engineered with mutated amino
acids that interacted with key amino acids of the RBDs, resulting in higher
binding affinity than human angiotensin-converting enzyme 2 (ACE2) and other
viral RBDs or hemagglutinins (HAs). The successful development of these Nbs
demonstrates the potential of molecular modeling as a low-cost and
time-efficient method for engineering effective Nbs against SARS-CoV-2. The
engineered Nbs have the potential to be employed in RBD-neutralizing assays,
facilitating the identification of novel treatment, prevention, and diagnostic
strategies against SARS-CoV-2.






[STEPS]
SECTION: 1. Validation of protein-protein docking server
1. Prepare the protein datasets consisting of 29 nanobody (Nbs) and 86 antibodies (Abs) complexed with RBDs from the Protein Data Bank (PDB) (https://www.rcsb.org/) for blind docking.

9. The similarity of amino acid sequences of 29 Nbs is analyzed using the Clustal Omega server (https://www.ebi.ac.uk/Tools/msa/clustalo/).
SECTION: 2. Selection of lead Nbs
7. The 29 Nbs are redocked with each targeted RBD using a blind docking method using HDOCK. 
12. Nb residues that had no interaction and repulsion with RBD are considered for the mutation process.

SECTION: 1. Validation of protein-protein docking server
2. Remove heteroatoms/molecules, including metal ions, small molecules, water molecules, and His-tags, from all complexes.

SECTION: 2. Selection of lead Nbs
8. Calculate the RMSD values to assess the accuracy of the docking poses of the 29 Nbs with respect to all targeted RBDs. Present the docking scores and RMSD values for each RBD in terms of the mean.
13. The AMBER ff14SB force field is applied for structural energy minimization before the docking process of mutated Nbs.

SECTION: 3. Structural-based engineering and broad specific binding of Nbs
11. To improve the binding affinity of Nbs to all targeted RBDs, the two lead Nbs are mutated using the site-direct mutagenesis feature on the Discovery Studio program.

SECTION: 1. Validation of protein-protein docking server
3. Prepare the protein chains of RBDs and ligands (Nbs or antibodies) separately using Discovery Studio software.

SECTION: 1. Validation of protein-protein docking server
4. The missing amino acids in the protein chain are remodeled using the SWISS-MODEL expert system (https://swissmodel.expasy.org/). 

SECTION: 1. Validation of protein-protein docking server
5. Perform blind docking using seven protein-protein docking programs including;
1) HDOCK  (http://hdock.phys.hust.edu.cn/)
2) ATTRACT (http://www.attract.ph.tum.de/services/ATTRACT/attract.html) 
3) pyDockWEB (https://life.bsc.es/pid/pydockweb)
4) GRAMM-X (http://vakser.compbio.ku.edu/resources/gramm/grammx/)
5) PatchDock (https://bioinfo3d.cs.tau.ac.il/PatchDock/) 
6) FRODOCK (http://frodock.chaconlab.org/) and
7) ZDOCK (https://zdock.umassmed.edu/).

SECTION: 1. Validation of protein-protein docking server
6. The root mean square deviation (RMSD) values of the ligands (Nb or Ab) are calculated using the Discovery Studio program. 

10. The lead Nbs are selected based on the best mean docking score, lowest RMSD, and diverse amino acid sequences, which are then employed for structure-based engineering.

SECTION: 4. Physicochemical properties prediction of engineered Nbs
16. The contact surface amino acids and chemical interactions are determined using the PDBsum server (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html).



14. Calculate the ΔHDOCK value, and choose the mutated residue at a specific position that exhibited the lowest ΔHDOCK for multi-point mutation.

15. To investigate the broad specific binding of engineered Nbs, cross-docking between Nb and all targeted RBDs, ACE2, and other viral RBDs/HAs is performed using the HDOCK program.



SECTION: 4. Physicochemical properties prediction of engineered Nbs
17. The physicochemical properties are predicted using the ProtParam (ExPASy) tool (https://web.expasy.org/protparam/). 

SECTION: 4. Physicochemical properties prediction of engineered Nbs
18. The PI value is calculated using the Protein–Sol web server (https://protein-sol.manchester.ac.uk/).



SECTION: 4. Physicochemical properties prediction of engineered Nbs
19. The total charge is calculated by PROTEIN CALCULATOR v3.4 (https://protcalc.sourceforge.net/).