Exploring the effect of ritonavir and TMC-310911 on SARS-CoV-2 and SARS-CoV main proteases: potential from a molecular perspective

Aim: As coronavirus (CoV) disease 2019-associated pneumonia spreads globally, there has been an urgent need to combat the spread and develop vaccines. Materials & methods: We used an integrated computational algorithm to explore the binding mechanism of TMC-310911/ritonavir (RVT) with SARS-CoV-2 and SARS-CoV main proteases. Results: RVT and TMC-310911 had favorable interactions with the proteases, and these high interactions are facilitated by some significant residues such as Asn133, Gly195 and Gln192. Our study further implicated two important rings in the structure of RVT as a possible chemical culprit in its therapeutic activity. Conclusion: Although there are conflicting clinical results on the therapeutic potency of RVT in the treatment of coronavirus disease 2019, our findings provided molecular insight into the binding mechanism of TMC-310911 and RVT with SARS-CoV-2 and SARS-CoV main proteases.

been reported to have helped patients overcome the infection through boosting their immune system [8]; however, currently, no vaccines or drugs are available for the treatment of this disease [11]. The global spread of COVID-19 and the absence of vaccines or drugs have necessitated the demand of drug repurposing. Crucial proteins that have gained attention in the development of potential COVID-19 drugs include SARS-CoV-2 main proteases (COV), RNA-dependent RNA polymerase, RNA binding N terminal domain of nucleocapsid protein, viral ion channel, 2 O-ribosemethyltransferase and human angiotensin-converting enzyme 2 receptor [12][13][14]. Several existing drugs such as remdesivir, chloroquine, hydroxychloroquine, camostatmesylate, lopinavir, etc., have been considered for targeting different phases of the virus life cycle.
Ritonavir (RVT) is an antiretroviral drug used in the treatment of advanced HIV, it is used as a protease inhibitor and also used to boost other protease inhibitors [15]. It is sometimes used in combination with antiretroviral drugs such as nucleotide reverse transcriptase inhibitor or nucleoside [15,16]. An in vivo study found a reduced risk of mortality and hypoxia in 41 SARS-CoV patients who were administered a combination of lopinavir/RVT and ribavirin, when compared with controls administered ribavirin alone [17,18]. A different study from Korea revealed that there was a significant reduction in CoV titers upon lopinavir-RVT administration [19]. Contradictory, a combination of lopinavir and RVT was investigated in a controlled trial study, patients with COVID-19 were administered either lopinavir-RVT 400/100 mg orally twice daily plus standard of care, or standard of care alone, there was no therapeutic benefit observed upon administration [20]. There are at least three randomized clinical trials currently been carried out to determine the therapeutic efficacy of a combination of lopinavir and RVT [21]. Similarly, ASC-09 also referred to as TMC-310911 is a protease inhibitor that is still under clinical studies to treat HIV infection. It has shown in vitro activity against the HIV strains that have developed resistance to other protease inhibitors [22]. There is currently an ongoing clinical trial that seeks to evaluate the safety and efficiency of a combination of ASC09/RVT on CoV infection (clinicaltrials.gov/ct2/show/NCT04261907) [23].
In this study, we repurposed two existing HIV-protease inhibitors RVT and TMC-310911 (ASC09) to target SARS-CoV-2 main protease (COV) and SARS-CoV main protease (SARS) to explore their possible mode of inhibition.

Structure preparation & dynamic studies
The starting structures of COV and SARS were obtained from Protein Data Bank with PDB ID 6LU7 [24] and 5N19 [25], respectively. Cocrystalized molecules identified with the proteins were deleted and the addition of missing residues was performed using modeler [26]. RVT and TMC-310911 were retrieved from PubChem, the 2D structures were converted to 3D structures and optimized using B3LYP/6-311++G(d,p) [27] level of Gaussian16 [28]. The final structures were saved as Mol2 files. Molecular docking of the proteases with the inhibitors was carried out using AutoDock Vina [29] inbuilt in UCSF chimera [30]. AutoDock Vina is software developed by O Trott of Molecular Graphics Laboratory at the Scripps Research Institute [29]. Validation of the docking results was carried out by redocking multiple times. COV and SARS were prepared for docking by the removal of water and cocrystallized ligands. Hydrogens were added and optimization of the hydrogen-bonding network was carried out with the aid of Avogadro software [31]. The clean COV and SARS structures were saved for docking. During the docking process, we allotted partial chargers called Gasteiger to the ligands. The AutoDock [32] GUI provided by the Molecular Graphics Laboratory tool was utilized to define the AutoDock atomic types [33]. The defined parameter grid dimensions used in docking TMC-310911 with COV and SARS were center (x = -17.56, Y = 0.29 and Z = -21.46) and size (X = 16.00, Y = 16.06 and Z = 13.19), while center (x = -17.19, Y = 0.44 and Z = -21.95) and size (X = 14.21, Y = 16.58 and Z = 11.86) were used for docking RVT with COV and SARS. The remaining values were set to default. Molecular dynamic simulation run was carried out with the aid of Amber 19 [34] software using the FF14SB force field [35]. The general Amber force field and restrained electrostatic potential were used in describing the atomic charges of RVT and TMC-310911. Leap variant present in Amber 19 was used for system neutralization and hydrogen atoms addition [36]. The system was solvated with an orthorhombic box of TIP3P water molecules surrounding all protein atoms at a distance of 9Å [27]. System minimization was carried out first with a 2000 step minimization utilizing a restraint potential of 500 kcal/mol. Second, we used a 10,000-step full minimization process without restraint. Afterwards, the system was gradually heated at a temperature of 0-300 k at 50 ps. The system solutes are kept at a potential harmonic restraint of 10 kcal mol -1Å-2 and collision frequency of 1.0 ps -1 . Next, the equilibration of 500 ps was carried out. The temperature and pressure were kept constant at 300 k and 1 bar (isobaric-isothermal ensemble, constant temperature and pressure using Berendsen barostat).  Each step of the simulation was run for 2 fs and an single-precision floating-point precision model was adopted. The simulations were kept at constant temperature and pressure, and Langevin thermostat at collision frequency of 1.0 ps -2 . Six systems were set up for molecular dynamics (MD) simulations. These systems are unbound COV, COV bound to TMC-310911 (COV TMC), COV bound to RVT (COV RVT), unbound SARS, SARS bound to TMC-310911 (SARS TMC) and SARS bound to RVT (SARS RVT). PTRAJ variant of Amber 19 was adopted for further analysis which included root mean square deviation (RMSD) and radius of gyration (RoG) [37]. The data plots were then made with ORIGIN analytical tool and visualization was done using UCSF Chimera [38].

Ritonavir & TMC-310911 binding free energy with COV & SARS
The molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) [39] was employed in the estimation of the binding strength in the COV TMC, COV RVT, SARS TMC and SARS RVT. MM/PBSA is described as an end-point energy estimation used in the calculation of binding mode of ligands and their corresponding protein target. MM/PBSA is described as: G gas represents the total gas-phase energy calculated by intermolecular energy ( E int ), electrostatic energy ( E elel ) and van der Waals energy ( E vdW ). G sol represents the solvation energy, T S represents entropy change.
G ele,sol(GB) describes polar desolvation energy while G np,sol describes the nonpolar desolvation energy. The γ is the surface tension proportionality constant and is set to 0.0072 kcal mol -1Å-2 , β is a constant equal to 0 and SASA is the solvent-accessible surface area (Å 2 ).

Mechanism of inhibition of ritonavir & TMC-310911 on SARS-CoV-2 & SARS CoV main proteases
RVT and TMC-310911 (TMC) like any other HIV protease inhibitors work by inhibiting proteases found in the liver, intestine and other places [40,41]. The binding of RVT to the active site of HIV proteases prevents cleavage of the viral polyproteins subsequently leading to inactive and noninfectious viral components [15]. Similarly, TMC-310911 works following this pharmacokinetic and pharmacodynamic route [22]. Therefore, we explored the mechanism of inhibition and structural perturbative effect of RVT and TMC-3410911 binding to COV and SARS. MM/PBSA has been widely employed in the drug development and computer-aided drug designs space, it estimates the binding strength between an inhibitor and a protein [42,43]. COV RVT, SARS RVT, COV TMC and SARS TMC had binding free energy of -29.46, -32.34, -32.29 and -47.19 kcal/mol, respectively. Findings from this analysis revealed that the RVT and TMC-310911 had a favorable binding interaction with the COV and SARS (Table 1). Furthermore, from Table 1, it is observed that the various free energy components facilitated this strong interaction. Most especially the electrostatic and van der Waals interactions observed in the gas phase of COV TMC and SARS TMC.

Energy (Kcal/mol)
We used a representative snapshot and active site residue decomposition to further explore the time-wise bond interaction occurring between major residues in the active site of COV/SARS and TMC-310911/RVT. In the COV RVT system, Asn133, Gly195 and Gln192 elicited hydrogen bond interactions, Phe185 formed π-sulphur interaction with the S atom present in RVT ring ( Figure 1A). The decomposition of this interaction also showed that favorable energy was contributed by residues possessing energy contributions greater than -0.5 kcal/mol. Ala194, which formed a pi-alkyl bond with RVT, contributed the highest total, van der Waal, and electrostatic energies to the overall binding ( Figure 1B). Decomposing the active site residues of the SARS RVT system revealed that Met49 and Gln189 contributed the most to the overall binding of RVT to SARS. In comparison to the COV RVT system, SARS RVT had higher G bind , this could be as a result of the high sigma bond formed by Pro168, Met49 and Cys145 ( Figure 1C). Binding of TMC-310911 to COV and SARS demonstrated an overall favorable binding when compared with RVT binding. This is evident by the high energy decomposition of the individual active site residues. Asn142 and Asp187 elicited a high hydrogen bond, while Ser46, Thr45 and Met 49 formed a pi-alkyl bond with TMC-310911 ( Figure 2).
To further explore the possible mechanism of the action of RVT on SARS-CoV-2 main protease, we examined the interaction trend of two important rings in RVT in the course of the simulation run. Interestingly, these two rings formed strong interaction with residues embedded inside the active site. These interactions sequestered RVT deeper into the active site (Figure 3).   Figure 4B).

Discussion
Sequence similarity is described as a measure of the empirical relationship that exists between sequences, it establishes the propensity of sequences evolving from a common ancestor. Joshi et al. have demonstrated that COV has higher sequence similarity with SARS when compared with MERS-CoV main protease [44]. In this study, RVT and TMC-310911 were repurposed to bind with COV and SARS. Although there are conflicting results on the therapeutic effectiveness of RVT and TMC-310911 in the treatment of COVID-19. For instance, in a clinical trial study, the combination of lopinavir and RVT was investigated in a controlled trial study, patients with COVID-19 were administered either lopinavir-RVT 400/100 mg orally twice daily plus standard of care or standard of care alone, there was no therapeutic benefit observed upon administration [20]. RMSD is a commonly used quantitative parameter employed to estimate the similarity between two superimposed structures. RMSD can be computed for different components of a biomolecule. In MD simulation, the RMSD is often calculated for the Cα of the entire protein structure, for example, those found in the loop, active site and perhaps transmembrane helices. Many types of researches have used RMSD as a measure of protein stability and equilibration. RMSF is defined as the measure of the atomic displacement of a single or a group of atoms relative to the starting or reference structures, averaged over the number of atoms [45]. RoG is a function used to define the distribution of atoms of a protein around its axis. The most significant parameter used in the prediction of protein compactness is RoG [46]. Insight from RMSD, RMSF   [47]. Upon binding of RVT to SARS-CoV-2 main protease, the structural compactness of the COV TMC, COV RVT system were lesser than that of the COV system, similar trend was observed in the SARS TMC and SARS RVT systems.

Conclusion
Presently, there is no available clinical antiviral compound (or drug) with therapeutic evidence that cures COVID-19 and several strategies are being considered to treat this disease, including repurposing drugs that are active against SARS-CoV and MERS-CoV. Other strategies such as inhibitors of viral and host protease, host-directed therapies and the use of antibodies are being currently explored. In this study, we explored the inhibitory effect of RVT and TMC-310911 on COV and SARS. Results from this study revealed that RVT and TMC-310911 had strong binding interaction with COV and SARS. Two rings were found to be crucial to the binding of RVT to COV and SARS. These two rings could be further explored in the development of inhibitors specifically tailored to target SARS-CoV-2 proteases.

Future perspective
We employed integrated computational algorithms and force field protein-ligand dynamics calculations to repurpose and explore the possible mechanism of inhibition of RVT and TMC-310911 upon binding to COV and SARS. Findings from this study can facilitate a new frontier in structure-based design of highly effective and tailored inhibitors of SARS-CoV-2 main protease in the treatment of CoV infections.

Summary points
• Upon ritonavir (RVT) binding to SARS coronavirus 2 (SARS-CoV-2) main protease (COV), Asn133, Gly195 and Gln192 elicited hydrogen bond interactions, while Phe185 formed π-sulphur interaction with the S atom present in the RVT ring. • RVT and TMC-310911 had strong interactions with the proteases, and these high interactions are facilitated by some significant residues found in their active site. • Despite having similar residues in their active sites, COV elicited ligand interactions with dissimilar residues as SARS-CoV protease. • Two rings were found to be crucial to the binding of RVT to COV and SARS-CoV main proteases.

Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Open access
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