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 nanobodies (Nbs) and 86 antibodies (Abs) complexed with RBDs from the Protein Data Bank (PDB) (https://www.rcsb.org/) for blind docking.
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: 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. Superimpose the docking pose with native to calculate the root mean square deviation (RMSD) values of the ligands (Nb or Ab) using the Discovery Studio program. 
SECTION: 2. Selection of lead Nbs
7. Redock all 29 Nbs listed in the S1 Table with the targeted RBDs using a blind docking method through the HDOCK server.

SECTION: 2. Selection of lead Nbs
8. Minimized the energy of the Nb/RBD complexes utilizing the AMBER ff14SB force field, providing the complexes with the lowest binding energy.

SECTION: 2. Selection of lead Nbs
9. For each Nb and RBD, a separate redocking was performed using HDOCK to determine the best docking score.

SECTION: 2. Selection of lead Nbs
10. Superimpose the docking pose with native to calculate the root mean square deviation (RMSD) values of the Nbs using the Discovery Studio program. Present the docking scores and RMSD values for each RBD in terms of the mean.
SECTION: 2. Selection of lead Nbs
11. 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
12. The lead Nbs are selected based on the best mean docking score, lowest mean RMSD, and diverse amino acid sequences, which are then employed for structure-based engineering.
SECTION: 3. Structural-based engineering and broad specific binding of Nbs
13. The Nb/RBD complexes in native form, in their initial state after blind docking using the HDOCK. 

SECTION: 3. Structural-based engineering and broad specific binding of Nbs
14. Minimized the energy of the Nb/RBD complexes utilizing the AMBER ff14SB force field, providing the complexes with the lowest binding energy. This was done to align the torsion angles of complementary amino acid side chains between Nb and RBD, ensuring uniformity in the lowest binding energy and preventing any atom that might have astride. 

SECTION: 3. Structural-based engineering and broad specific binding of Nbs
15. Redock the optimized Nb and RBD yielding the native form's score as the initial score (HDOCKNb (native)).


SECTION: 3. Structural-based engineering and broad specific binding of Nbs
16. Mutate the amino acid on crucial target residues at the interaction site of Nbs to each target RBD,
while considering the non-contact/interaction amino acid(s) and amino acid(s) that caused unfavorable interactions.

SECTION: 3. Structural-based engineering and broad specific binding of Nbs
17. Prior to redocking, each mutated amino acid was subjected to optimization using the CHARM force field within the Discovery Studio program, resulting in the post-mutation score as HDOCKNb (mutant). 

SECTION: 3. Structural-based engineering and broad specific binding of Nbs
18. Calculate the ΔHDOCK value, and choose the mutated residue at a specific position that exhibited the lowest ΔHDOCK for multi-point mutation.
SECTION: 3. Structural-based engineering and broad specific binding of Nbs
19. 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 as same this procedure from 13 to 15.
SECTION: 4. Physicochemical properties prediction of engineered Nbs
20. 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).
SECTION: 4. Physicochemical properties prediction of engineered Nbs
21. The physicochemical properties are predicted using the ProtParam (ExPASy) tool (https://web.expasy.org/protparam/). 
SECTION: 4. Physicochemical properties prediction of engineered Nbs
22. 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
23. The total charge is calculated by PROTEIN CALCULATOR v3.4 (https://protcalc.sourceforge.net/).