Selocalcitol: Scope of inhalational formulation for prevention and cure of COVID-19 illness

Vitamin D has generated interest in the context of COVID-19 pandemic. Some authorities believe that it is benecial; others think that without proven deciency it should not be administered. The results of clinical trial are still awaited. Therefore nothing regarding the matter is conclusively known. In this context we believe that selocalcitol, a Vitamin D analogue if administered via inhalational route, has the potential to prevent SARS-CoV2 infection. The basis of this idea is presented here.

[2] The disease is named as COVID-19. [2] It ranges from asymptomatic illness to a severe form of the disease. [5][6] The death toll is considerably high, and at the present moment apart from the Russian vaccine, there is no approved medication or vaccine for prevention or cure of COVID-19 illness. [7][8] Mankind has masked its face and maintaining distance to prevent viral transmission. [9] SARS-CoV2 is recognized as a respiratory pathogen. The viral S-protein binds to Angiotensin-converting receptor 2 located at bronchial epithelial cells. [10][11] After binding the ACE-2 is downregulated and then virus enters the host cell. [10] ACE2 is an isoform of Angiotensin-Converting Enzyme(ACE). [12] ACE catalyzes the formation of angiotensin II from angiotensin I. [12] On the other hand ACE2 catalyzes the breakdown of Angiotensin I to bene cial peptides. [13][14] The virus binding alters the ratio of ACE to ACE2. [15] Following virus binding, ACE2 function becomes less compared to ACE function. [15] Therefore, angiotensin II formation may increase. [14][15] Angiotensin II is a pulmonary vasoconstrictor. [16] So, the COVID-19 illness can generate severe respiratory symptoms leading to fatality. [17][18][19] There is considerable current interest in Vitamin D status and COVID-19 illness. [20][21] Many authorities believe that vitamin D de ciency is related to COVID-19 illness. [22][23] We believe that Vitamin D has a paradoxical relationship with COVID-19 illness. Vitamin D inhibits renin to form less angiotensin I (the precursor of angiotensin II). [24][25] So, Vitamin D indirectly inhibits the formation of angiotensin II. [26] Vitamin D inhibits ACE as well. [27] Further Vitamin D expresses ACE2 in host cells. [28] This also helps to overcome the ill effects of inhibition of ACE2 function due to virus binding. Nevertheless, vitamin D induced overexpression of ACE2 will also invite the viral infection as the virus binds to ACE2 prior entry to the host cell. Therefore, using tools of computational biology we have searched some vitamin D analogues that bind better with VDR and ACE2 in comparison to Vitamin D. Such molecule is expected to overexpress ACE2, but the virus will not get chance to bind with the protein as the analogue will preoccupy the virus binding site. The screened analogue and vitamin D were docked with VDR and ACE2 in separate runs for 6 times using AUTODOCK 4.2.6 and GOLD 5.3.0 software to obtain free energy of binding and the binding score. The interaction of the analogue at a known ligand-binding site is validated by Molecular Dynamics simulation run for 10 ns using GROMACS 2018.4 software following the instruction of the software manufacturer. Lys31 of ACE2 is essential for binding to SARS-CoV2. In VDR Tyr 143 and His305 are critical for ligand binding. [29][30][31][32][33] The NZ atom of Lys31 from ACE2 and OH & NE2 of Tyr143 & His305 from VDR were chosen as the grid centre for docking.
The docked structures exhibiting the highest negative binding energy and best GOLD score with ACE2 was considered for MD simulation. In the case of VDR, docked structures at His305 were chosen. Drug topology les were generated using the SwissParam tool, and CHARMM 27 force-eld was used throughout the simulation. The Protein-drug complexes were solvated in 1.2nm cubic boxes of water using the TIP3P water model, and neutralization was performed using required sodium ions (Na+).
Energy minimization was performed for 50,000 steps using the steepest descent method. The system was then equilibrated using NVT and NPT protocols for 50,000 steps each. Then the entire system was considered for MD simulation of 10ns runs at 300K temperature and 1 bar pressure and results were visualized using Pymol. Xmgrace plotting tool was used to analyze the graphs of potential energy, root mean square deviation (RMSD), radius of gyration, hydrogen bonds, and other parameters as discussed in the results.

Results
In the screening study, Selocalcitol has shown most favourable binding energy with Lys31 of ACE2 compared to 25-hydroxy cholecalciferol (Vitamin D) or other studied Vitamin D analogues (Table 1).
Selocalcitol has shown signi cantly more (p<0.0001) favourable binding free energy compared to Vitamin D when docked with ACE2 (n=60) ( Table 2). When the same in-silico experiment is repeated using GOLD software, the analogue has shown signi cantly (p<0.0001) better binding score (or Gold score) when compared to Vitamin D. The above observation is true for VDR when the grid centre of docking was His305 in both Autodock and GOLD. However, when Tyr143 was considered as grid centre, the similar pattern was observed with GOLD. The mean ± SD of the observed binding energy values and GOLD score are given in Table 2 & 3. Binding orientations of the Vitamin D and Selocalcitol molecules after docking with ACE2 and VDR are shown in Figure 1 & 2. In all the protein-drug complexes, at least one hydrogen bond is observed. In addition to the hydrogen bond(s), non-bonded interactions were also visualized.
The MD simulation run of the docked complex of ACE2 bound with selocalcitol in autodock showed stable binding. The potential and kinetic energy of the complex were stable throughout the simulation run. The radius of gyration decreases with time, rmsd stabilizes after 5 ns run, and on average one hydrogen bond is observed throughout the run (Figure 3). Similar results were obtained when docked pdb le generated using GOLD was used for MD simulation (Figure 4).
In the case of VDR, Selocalcitol docked at His305 site was used for MD simulation. At VDR as well, selocalcitol has shown stable interaction throughout the run time ( Figure 5

Discussion
Authorities believe that Vitamin D is bene cial for the management of COVID-19 illness. [34]. Opposing views are also present. [35] Amidst these two divergent views, we believe that Vitamin D exhibits a paradoxical relation with COVID-19 illness. [20] Vitamin D inhibits renin. It will reduce the production of angiotensin II. It is going to be bene cial. On the other hand, Vitamin D expresses ACE2. It will cause SARS -CoV 2 infection as the virus binds with ACE2. Therefore, Vitamin D analogues may be bene cial than Vitamin D per se in COVID-19 illness if it inhibits renin and simultaneously preoccupies the virus binding site at ACE2. It is in this context our observed results are important.
We have predicted that selocalcitol binds with VDR and ACE2 better than Vitamin D. Vitamin D binding with VDR inhibits renin gene expression. [36][37]24] We have also predicted that selocalcitol binds with VDR better than vitamin D. This is worth validating experimentally. If our prediction comes to be true by experimental studies then; selocalcitol will inhibit renin action better than vitamin D.
Further, in-silico selocalcitol has occupied the virus binding site of ACE2 (i.e. Lys31) better than vitamin D. So, selocalcitol may inhibit the virus binding with the ACE 2. SARS-CoV 2 binding with ACE2 is critical for the COVID-19 illness. So selocalcitol if blocks the virus binding site is expected to prevent virus binding if supplied before the virus exposure. Additionally, inhibition of the renin expression is expected to reduce pulmonary complications of COVID-19 illness. Based on the above reasons we strongly recommend experimental veri cation of our prediction, and if proved to be valid, we believe that an inhalational formulation of selocalcitol can be thought as a vaccination strategy for prevention of COVID-