Molecular and Structural Aspects of Clinically Relevant Mutations of SARS-CoV-2 RNA-Dependent RNA Polymerase in Remdesivir-Treated Patients
Carmen Gratteri, Francesca Alessandra Ambrosio, Antonio Lupia, Federica Moraca, Bruno Catalanotti, Giosuè Costa, Maria Bellocchi, Luca Carioti, Romina Salpini, Francesca Ceccherini-Silberstein, Simone La Frazia, Vincenzo Malagnino, Loredana Sarmati, Valentina Svicher, Sharon Bryant, Anna Artese, Stefano Alcaro
Pharmaceuticals, doi:10.3390/ph16081143
1) Background: SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target to fight COVID-19, and many RdRp inhibitors nucleotide/nucleoside analogs, such as remdesivir, have been identified or are in clinical studies. However, the appearance of resistant mutations could reduce their efficacy. In the present work, we structurally evaluated the impact of RdRp mutations found at baseline in 39 patients treated with remdesivir and associated with a different degree of antiviral response in vivo. (2) Methods: A refined bioinformatics approach was applied to assign SARS-CoV-2 clade and lineage, and to define RdRp mutational profiles. In line with such a method, the same mutations were built and analyzed by combining docking and thermodynamics evaluations with both molecular dynamics and representative pharmacophore models. (3) Results: Clinical studies revealed that patients bearing the most prevalent triple mutant P323L+671S+M899I, which was present in 41% of patients, or the more complex mutational profile P323L+G671S+L838I+D738Y+K91E, which was found with a prevalence of 2.6%, showed a delayed reduced response to remdesivir, as confirmed by the increase in SARS-CoV-2 viral load and by a reduced theoretical binding affinity versus RdRp (∆Gbind WT = -122.70 kcal/mol; ∆Gbind P323L+671S+M899I = -84.78 kcal/mol; ∆Gbind P323L+G671S+L838I+D738Y+K91E = -96.74 kcal/mol). Combined computational approaches helped to rationalize such clinical observations, offering a mechanistic understanding of the allosteric effects of mutants on the global motions of the viral RNA synthesis machine and in the changes of the interactions patterns of remdesivir during its binding.
Principal Component Analysis (PCA) Functionally relevant slow motions were analyzed by means of PCA. Specifically, diagonalization of the covariance matrix was performed using the gmx covar tool of GROMACS on the backbone atoms of both the Nsp12/Nsp8/Nsp7/RdRp/NiRAN and the RNA of the WT, 3M 2 , and 5M systems to compare the effect of the mutants with respect to the WT. Then, the motion of the protein was identified by projecting the first four eigenvectors with the gmx anaeig module. Graphs were plotted using the Gnuplot tool.
Pharmacophore Model Generation along the MDs The MDs trajectories, strided every 1 ns, of the three mutated systems (WT, 3M 2 , and 5M), complexed to remdesivir, were used to create representative pharmacophore models (RPMs). RPMs were extracted to understand the dynamic behavior and the chemical features of remdesivir in all of the systems (200 frames for each simulation). This is an innovative technique to reveal the interaction pattern of a ligand in the binding pocket. The advanced version of LigandScout 4.4.9 software [33] was used for the extended pharmacophore investigation on the trajectories of the MDs.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph16081143/s1 : Figure S1 : flowchart schematically representing the overall approach adopted in the study. Figure S2 : RMSF plot built on the four PCs revealing the changes in the fluctuations of 3M 2 and 5M..
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'abstract': '<jats:p>(1) Background: SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising '
'therapeutic target to fight COVID-19, and many RdRp inhibitors nucleotide/nucleoside analogs, '
'such as remdesivir, have been identified or are in clinical studies. However, the appearance '
'of resistant mutations could reduce their efficacy. In the present work, we structurally '
'evaluated the impact of RdRp mutations found at baseline in 39 patients treated with '
'remdesivir and associated with a different degree of antiviral response in vivo. (2) Methods: '
'A refined bioinformatics approach was applied to assign SARS-CoV-2 clade and lineage, and to '
'define RdRp mutational profiles. In line with such a method, the same mutations were built '
'and analyzed by combining docking and thermodynamics evaluations with both molecular dynamics '
'and representative pharmacophore models. (3) Results: Clinical studies revealed that patients '
'bearing the most prevalent triple mutant P323L+671S+M899I, which was present in 41% of '
'patients, or the more complex mutational profile P323L+G671S+L838I+D738Y+K91E, which was '
'found with a prevalence of 2.6%, showed a delayed reduced response to remdesivir, as '
'confirmed by the increase in SARS-CoV-2 viral load and by a reduced theoretical binding '
'affinity versus RdRp (ΔGbindWT = −122.70 kcal/mol; ΔGbindP323L+671S+M899I = −84.78 kcal/mol; '
'ΔGbindP323L+G671S+L838I+D738Y+K91E = −96.74 kcal/mol). Combined computational approaches '
'helped to rationalize such clinical observations, offering a mechanistic understanding of the '
'allosteric effects of mutants on the global motions of the viral RNA synthesis machine and in '
'the changes of the interactions patterns of remdesivir during its binding.</jats:p>',
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