The fluctuation of a group of atoms and the flexibilities of different regions of protein were analyzed from RMSF calculation. In case of protein only, the higher fluctuation was observed for the Glu47 residue (0.27 nm) in domain I and for Tyr154 residue (0.24 nm) in domain II, but the rest of residues in domains I and II had low fluctuation below 0.2 nm. In the case of protein-hit complexes, the conformational fluctuation was higher at the domain III as compared to the domains I and II due to binding of the hits in between domains I and II. The average RMSF value of the native protein was found to be 0.119 nm, whether the protein-inhibitor complexes displayed lower fluctuations than M Pro displaying the stabilities of the complexes after binding (Table , Figure ). Moreover, the detailed analysis of RMSF fluctuations of different residues and domains (I, II, III, and loop) were carried out as shown in supplementary Table . The M Pro -famotidine complex displayed higher RMSF fluctuation from 46 to 52 residue at domain I while MET49 displayed 0.48 nm RMSF value that was the highest in this region. In The M Pro -famotidine complex, domain II residues between 153 to 155 displayed RMSF value between 0.15 nm to 0.21 nm which was the highest fluctuation observed in domain II. The alkaloid CNP0416047 displayed favorable non-bonding interactions with the different residues like HIS41, HIS64, Gly143, SER144, CYS145, HIS164, GLU166, ASP187, ARG188, GLN189, GLN192, THR190, MET49, MET165, and LEU27 as evident by the low RMSF of these residues compared to the unligated M Pro . A greater fluctuation was observed in the region of 47-53 residues of domain I due to hydrophobic interaction caused by tyrosine moiety attached to C6 of quinazoline ring of CNP0416047. The 3-hydroxy anisotine interacted with the M Pro with different amino acid residues like HIS164, GLY143, ASP187, ASN142, SER144, ARG188, GLN189, THR26, LEU141, HIS41, GLU166, CYS145, CYS44, TYR54, MET165 during the entire simulation run as evident by low RMSF values compared to unligated M Pro . Different binding residues of the anisotine with M Pro during MD simulation (SER46, TYR54, GLU189, GLY143, SER144, ASN142, ARG188, HIS164, ASP187 etc.) displayed comparable RMSF to the unligated M Pro . Overall the domain II region was stabilized after the binding of inhibitors as evident by lessening of average RMSF compared to unligated M Pro .