Protein structure
The outbreaks of virus attack at Wuhan region of China is the case of new type of coronavirus from bat origin, 2019-nCoV [4]. For this reason, we have very little information about its genetic material as well as structures of different proteins. Fortunately, in recent time, structure of CoV Mpro protein of 2019-nCoV has been determined using x-ray crystallography [18]. It is interesting to observe that the amino acid sequence of CoV Mpro protein of 2019-nCoV is highly different from the previously known CoV Mpro. This mutation produce a novel variety of the protein with same activities. The change in the secondary and tertiary structure of the protein is clearly noticeable in the overlying picture of mutant (pink, pdb id: 6lu7) and one of the known protein [19] (cyan; pdb id: 2q6f) is shown below (Figure 1a). The huge change of the mutant protein in almost every region of it, is key cause for the drug resistance. It creates the scope of development of new compounds against 2019-nCoV.
The study has been extended further to identify the extent of mutation of the novel CoV Mpro by alignment of multiple protein sequence (Figure 1b). In this bioinformatics study, more two formerly known CoV Mpro (pdb ids: 2q6d and 2h2z) included to get more clear idea about the new mutation. Taking 2q6d protein as a reference sequence, it was found that the aligning of the length of mutant protein (6lu7) is covered almost 96% which is also found other two aproteins. The mutant protein exhibited consensus 70% which is minimum among them. This is the indication of huge mutation (Figure S1).
Choice of organic compounds
The mutant CoV Mpro contain nucleophilic amino acid residues (His-41, 163, 164 and 172) at the active site which can be utilized as nucleophile donor in Michael reaction (Figure S2). In natural sources, there are a number of heterocyclic moieties but the flavone (1) have a special recognition due to their easy availability and high activities on different health benefit [20]. 3- Benzylidene-4-chromanones (2) has high structural resemblance with flavone (Figure 2). In both the cases, there are one a,b-unsaturated C=C double bond and one rotatable phenyl ring is present. It can interact with different types of proteins and receptors as a blocking agent like flavones.
In the case 2, it contain one reactive exo-cyclic a,b-unsaturated C=C double bond without conjugation with any oxygen atom. The extended conjugation of ring oxygen with ketone group reduces the reactivity of the C2 (Michael acceptor site) and C4 position of the molecule 1. The presence of exo-cyclic C=C double bond at C3 without previous type of conjugation, allow the compound 2 for Michael reaction at C11 Therefore, there is chance ofcompound 2 as Michel acceptor during interaction with nucleophilic amino acid residue (Cys, Lys, His, Ser and Tyr) of protein molecule. For this reason we prefer to choose compound 2 in this study. Compound 2e-i were also be considered to explore the electronic features of 2 and it is discussed later.
Molecular Electrostatic Potential (MEP) Analysis
Molecular electrostatic potential surface give information about the electron rich and electron deficient parts of a molecule which is generate due to the presence of atoms with different electronegativity. This molecular detail helps to predict how much they are potential to take part in chemical reaction and to realize their mechanism of interactions. The high potential energy regions are electron poor (blue) and the low potential parts having correspondingly low potential energy (Red) and the neutral region is green [21].
The MEP of compounds 2a-i shows that these molecules are capable to form at least two hydrogen bonding (red paches) during protein binding with the help of ketone and chromanone ring oxygen except 2g and 2h (Figure 3). The electron density of chromanone aromatic ring is quite higher (yellow) due to conjugation of ring hetero-atom which is favourable for π-staking interaction with electron deficient amino acid residue. It is possible to modulate the electron density at benzylidene ring and C11 position by adding different functional groups at benzylidene part. Incorporation of oxygen containing functional groups at benzylidene part (2b, 2e and 2h) increases the electron density to the ring (Figure 3). The halogen atoms at this part are unable to influences the electronic behaviour of the ring (green for 2c, 2d and 2g) whereas the atom concentrate electron density around it. Therefore, it is expected that these compound may interact strongly with protein through π-staking as well as halogen bonding. In the case of compound 2f, the electron rich oxygen containing furan ring can bind strongly. The extended conjugated hydrophobic benzylidene system is 2i and sufficiently electron rich (Figure 3). Therefore, small red, yellow and blue patches on large green surface of compounds chosen for these studies are the indication of balance between hydrophilic and hydrophobic part which is essential for good binding to the protein.
For the assessment of bioavailability, absorption, distribution, metabolism and excretion (ADME) properties and pharmacokinetic studies of small molecules are major concern. To judge the ability of synthetic of isolated molecules as a drug and approval for clinical trials, it should fulfil the ADME requirements [22]. Here, ten molecules has been assessed for their bioavailability.
Lipophilicity
The standard descriptor for Lipophilicity is the partition coefficient between n-octanol and water (log Po/w). The partition coefficient is so important for pharmacokinetics drug discovery that SwissADME, a very useful web tool for pharmacokinetic evaluation provided a section for the property [23,24]. Several computational methods for log Po/w estimation have been developed with varying performances on different chemical sets. Normally multiple predictors have been used to opt for either the most precise method for a given chemical series or to know the consensus evaluation. The models should be diverse enough so that the prediction of the consensus log Po/w will be more accurate [25]. SwissADME provides access to five free predictive models in this connection, i.e. XLOGP3, an atomic method with corrective factors and knowledge-based library [26], WLOGP, a purely atomistic method based upon Wildman and Crippen's fragmentation method [27], MLOGP is an archetypal topology tool based on a linear relation with 13 molecular descriptors [28,29] SILICOS-IT, a 27 fragments, and seven topological descriptors hybrid system [30] and iLOGP is the physics-based method that uses free solvation energies of n-octanol and water which was calculated using the Generalized- Born and solvent accessible surface area (GB/SA) model.
Each log Po/w values for all the compounds are following same trends. The values are significantly below in the case of 2i which may be due to the enhancement of electron density and polarity in benzylidene part. The negligible change in the log Po/w values for the compound 2a and 2b indicates that the methoxy group cannot increase polarity of these molecules. The presence of halogens (Cl and Br) boost their lipid solubility (Figure 4). In case of 2c and 2g molecules, the changes are very high which is manifestation of the decrease in polarity and increase of the lipophilicity of the molecule for sulphur atom replacement. In this study, log Po/w values for designed compounds were found in the range of +2.29 to +4.25 (Figure 4, Table S1). These positive values signifies that all the molecules are highly lipophilic and fulfil the essential criteria for drug molecules.
Water solubility
Water solubility is an important requirement for any drug molecule intends to administer through the oral or parenteral route as the sufficient quantity of the active pharmaceutical ingredients has to be given in a small volume [31, 32]. SwissADME furnishes three different topological methods for estimating water solubility-Esol model [33], Ali model [34] and SILICOS-IT [35]. The Log S values of our compounds were in the range of -3.19 to -4.85 using ESOL Log S method, -2.9 to -4.41 using Ali Log S method and -4.25 to -6.38 for SILICOS-IT method, which are mentioned in the Table-1 and S2. Considering all the models we can conclude that the most of the compounds are moderately water-soluble and some of them show good solubility as the reference value of Log S for moderate solubility is between -4 to -6 and -2 to -4 for good solubility. This suggests that the molecules may be administered in the body through an oral or parenteral route.
Pharmacokinetics
ADME studies of these molecules also give place to these molecules in the approved drug molecule section. All these molecules have high gastrointestinal absorption which allow them for oral dosing and passed the most important and decision-making standard of drug discovery. Blood brain barrier (BBB) permeability test is passed by all of the compounds. Therefore, they are qualified a fundamental index of drug distribution. High negative values of skin permeability is also offers their less skin permeation. Non-inhibition of CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4 enzymes was found for most of the compounds (Table S2 and S3). Hence, the chance of degradation of these compounds inside the cell is slow and available for blocking of CoV Mpro. Bioavailability score of all the compounds is indication of high potential as a drug molecules. Synthesis of this compounds is very easy as stipulated in the synthetic accessibility of the compounds. All these values of above mentioned parameters is in the region found for the successful drug molecule. This is make it very close to drug like molecules.
Table 1: Predicted data of water solubility, pharmacokinetics, drug likeness and medicinal chemistry of the designed compound 2a-j
|
2a
|
2b
|
2c
|
2d
|
2e
|
2f
|
2g
|
2h
|
2i
|
2j
|
ESOL Log S
|
-3.83
|
-3.89
|
-4.41
|
-4.73
|
-3.92
|
-3.19
|
-4.85
|
-4.1
|
-4.29
|
-3.62
|
Ali Log S
|
-3.56
|
-3.73
|
-4.21
|
-4.29
|
-3.76
|
-2.90
|
-5.11
|
-4.01
|
-4.23
|
-4.41
|
Silicos-IT
LogSw
|
-5.41
|
-5.53
|
-6.02
|
-6.24
|
-5.15
|
-4.62
|
-6.38
|
-5.8
|
-5.5
|
-4.25
|
GI absorption
|
High
|
High
|
High
|
High
|
High
|
High
|
High
|
High
|
High
|
High
|
BBB permeant
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
Pgp substrate
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes
|
log Kp (cm/s)
|
-5.38
|
-5.57
|
-5.14
|
-5.36
|
-5.77
|
-5.95
|
-4.85
|
-5.16
|
-5.07
|
-6.37
|
Bioavailability
Score
|
0.55
|
0.55
|
0.55
|
0.55
|
0.55
|
0.55
|
0.55
|
0.55
|
0.55
|
0.55
|
Synthetic
Accessibility
|
2.74
|
2.87
|
2.73
|
2.76
|
3.00
|
2.77
|
3.17
|
2.55
|
3.05
|
3.29
|
Docking Study
Combination of understanding on bioinformatics and molecular docking are also being applied to forecast possible uses and production by industry [20]. Molecular docking has been used here to predict the inhibitory activity of compound 2a-j against mutant coronavirus main protease enzyme. In this study, we docked all the nine compounds with the CoV Mpro of novel (pdb id: 6lu7) as well as three previously known coronavirus (pdb ids: 2q6f, 2q6d and 2h2z). The crystal structure of mutant CoV Mpro contain a peptide fragment in its active site. The peptide also docked and the docking score is considered as the control. All the designed compounds shows much higher binding affinity toward CoV Mpro proteins with respect to the peptide (Table 2). It is so interesting that the compounds bind strongly with the new CoV Mpro in comparison with previously known proteins (Table 2). The study shows that the compound 2g perform best as anti- mutant CoV Mpro (Table 2). The more curious fact that the more lipophilic molecule bind more strongly with the proteins. The presence of halogen atoms increases electron density as well as hydrophobicity of the molecule which goes in favour of their strong binding. The compound exhibited excellent results in all the bioavailability studies as discussed previous sections.
Table 2: Docking score of designed compounds 2a-j and the peptide crystallised with mutant CoV Mpro
Compounds
|
6lu7
|
2q6f
|
2q6d
|
2h2z
|
Peptide
|
-4.47
|
-
|
-
|
-
|
2a
|
-7.16
|
-6.78
|
-5.16
|
-6.86
|
2b
|
-7.34
|
-6.56
|
-5.01
|
-6.37
|
2c
|
-7.58
|
-6.92
|
-5.56
|
-5.23
|
2d
|
-7.64
|
-7.01
|
-5.87
|
-5.29
|
2e
|
-7.58
|
-6.91
|
-5.81
|
-6.17
|
2f
|
-6.53
|
-6.23
|
-5.53
|
-6.09
|
2g
|
-7.85
|
-7.15
|
-6.19
|
-6.91
|
2h
|
-7.55
|
-6.89
|
-5.83
|
-6.24
|
2i
|
-7.23
|
-6.25
|
-5.12
|
-5.87
|
2j
|
-7.88
|
-7.21
|
-6.31
|
-7.13
|
All the compounds are docked inside the active site of the each of the CoV Mpro proteins (Figure 5a) by means of the hydrophobic as well as hydrogen bonding interactions (Figure S3-S10). The reddest patch in the electrostatic potential map (Figure 2) was on the ketone oxygen, forms hydrogen bonding with Glu 166 amino acid residue, observed in docking study. Amino acid residue Tyr 56, His 41 and 163 involved in π-staking interaction with both phenyl ring separately. Lone pair π-staking between Met 49 and 165 with Cl containing phenyl ring is also an interesting case. The chlorine is in hydrophobic interaction with Cys 44, Met 49, Pro 52 and Tyr 54 residues. The π-C-H interaction between His 163 and thiochromanone benzene ring as well as CH2 hydrogen and His 41 also found (Figure 5b).
The study provides an opportunity to modify these designed drug like compounds in its next level. There is a possibility of rotation of C-C bond designated by cyan colour in Figure 5b.
This rotation of bond can bring the ball shaped hydrogen at benzylidene ring closer to the Arg 188. Hydroxyl group substitution at the position of these hydrogen atoms can involve in hydrogen bonding with the amino acid residue. The ball shaped hydrogen in thiochromanone ring is closer to Phe 140 and carboxyl group of Glu 166. Replacement of this by NH2 groupmay capable for hydrogen bonding and ionic interactions. If these substitution occur, the compound 2j can come into existence. All the above studies also performed for the 2j and found that the compound performed like other except BBB permeability test. The MEP of 2j display that the electron density increase in both the aromatic ring due to electron donation of OH and NH2 groups (Figure 5c) which is favourable for π-staking interactions. The appearancedark blue regions in this MEP surface is clue for strong hydrogen bonding. The compound 2j bind slightly more strong way with CoV Mpro protein than that of 2g. The slight increment in docking score is for the compensation of the loss of huge number of interactions by the elimination of Cl group by the stabilising hydrogen bonging with OH and amine groups. Inside the active site, the attached groups (2j) mentioned earlier form expected hydrogen bonding interactions (Figure 5d).