3.1 Screening of phytochemicals against the active site of SARS-CoV-2 3CL pro
Structure- based virtual screening attempts to predict the best mode of interaction between two molecules to form a stable complex, and it uses scoring functions to estimate the force of non-covalent interactions between a ligand and molecular target. This technique has been used widely to identify potential inhibitors of SARS-CoV-2 replication [16, 49]. The results of virtual screening of phytochemicals from Vernonia amygdalina and Occinum gratissimum against the 3-chymotrypsin-like protease (3CLpro) of the novel SARS-CoV-2 alongside with the reference inhibitors (lopinavir and ritonavir) is represented in Table S1 (supplementary material). From the results, a hit list of 21 phytochemicals (Table S2) were selected based on their orientation at the catalytic site, the interacting residues and binding affinities comparable to those of reference inhibitors, lopinavir (∆G = -7.2 Kcal/mol) and ritonavir (∆G = -7.2 Kcal/mol).
Further binding docking of the topmost 10 compounds (Table 2) against the active regions of the target protein in SARS-CoV and MERS-CoV (Table 1), revealed that, these chemical structures (Table 2) had considerable docking scores (Table 3) and interactions with the coronavirus strains. Early homology models of SARS-CoV-2 3CLpro indicated close structural relation to those of other coronaviruses. Superimposition of the X-ray crystal structures of the 3CLpro of SARS-CoV-2, SARS-CoV and MERS-CoV indicates a considerable degree of structural similarity and conservation of the active site [8]. This is currently exploited for the development of SARS-CoV-2 3CLpro inhibitors that were based on previous compounds targeting the 3CLpro of SARS-CoV or MERS-CoV [8].
Table 2: Structures of reference inhibitors and top docked phytochemicals with the active sites of 3-Chymotrypsin-like proteases of Coronaviruses
S/N
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Compounds
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Class of compounds
|
Chemical Structure
|
Source Plants
|
S1
|
Lopinavir
|
|
![](data:image/png;base64,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)
|
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S2
|
Ritonavir
|
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![](data:image/png;base64,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)
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1
|
Vernolide
|
Sesquiterpene
lactones
|
![](data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAH0AAAB0CAYAAACojz3tAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAAXNSURBVHhe7Z2Nkdw2DIWvADeQBtyAG0gFqSAdpIO0kBZcg5tIF2km2ec7eGCE4J9IkBLfN4M5iRQBEm+plbjavTdCCGni34+/5CAo+oFQ9AOh6AdC0Q+Eoh8IRT8Qin4gFP1AKPqBUPQDoegHQtEPJFr0X1/2x8t+f9kvKCDxRIn+6WV/v+yfl0H0v16G2L+9jAQTJTpE/vq++YMvL0N8zvggkGyx2WCWI87n73s/gxfCn++bZBYpoWcLLzM6BU713943yWhSYgMp9+qvIP4o+gJSCfdE9spbsD54eg/EEzBVZvHalvDapC7k8CLA8byQG4AnmFeeo7aNd5yU8ZZtErnEp8pb8Nq3xuTizCBaE9+L9uf59spJADMTnxOcLMATZAYSJzImUaxIPMU+EAp+IBT9QO4iOtYIsBScWhEcDeIgnga3phGxQ7iD6FjiRT+xvo+FIdhMARALwmse9fnC7qIj2VgF1ItAKEO/7WysBW1z46boi0H/sAJowQsBK4Kt6PFiOzV+lFH0RSDxXv96RPB8oVzXYRsvKMQXw+cMFD2AkaLXjBPHiOFMAv9iuI6g6AHgfRz9S32og5kHq6F1jCK8bveo0/vuINFIuEYe6MCZAOREzdUJVmDrG0bRA5GHNDCrIQQ+u8ep1s5yEUdj9y3SRhvAXxFd0PW3BFfDeM+SgSCBvbc/EeD0jnt1zDQ8uVP70EZOJBm7NYA4dh0AMWW9QI5LEbmIVA0ER6clcUgoEokXwZPICSPCifWQahu9iFQNOmJnCl6dqfKdkCSnkm3R9Xa7pn0PeM8fvYg0BHl/THUCr1L70OMsepIuYpXapupr214BvkcuIg0D7zXewCOuTnXi9XYtpTapOt2m1L6X1XnNsmqm55LdIkKLn9KxXl0PW4sOvPduWz4iMbU+rh5ny2p8gdrjSoxaRJpGzdU76q210tsm1y5Vr/dL7VP0tEmB2YxZrcEZFb7tPf8SSvfpUm6thpZjPTwftly2veNbuOpD3jqRS4iMSYWz5/JZ3oMko5SUUn0P1qfel21dP4IrPnHW7FlE2hadZJ0Uuz8D8a//RsTsjTG7b0uQhEQOTseMjttKZP9CeezAErSOlaIHg37Zq+Wr98oU/QOK7kPRg6HoE6HoPhQ9GPQrZRR9ALsODP3iTJ8ERffZKjcYuH18B0uFWDpshaL7bJWbkQnZTXTpz8gxCq1j3So3IxOy08Bm94Wif7DLwCL6cXvR8Tk6RBbDZ75W9JpO7zCwqD60xtkhNz9AZ0rfxsQx1lJ45VFExm+NtTo3P4HOlE7vOMYzjd2PJDp2a7yVufkf6EztezqOLdkKUnFn96XV/6rcJEFnakXPAT9ikeRizupLj9/ovGSB4PaZdzzX1fp9Kz0obM8epI3hxfTKe7jia1QftsEb0JUkeZR8evWldjl62kqbnrbbUzOgEYNuTZ53fI+fGsRvq//b0TK4K8m4kkSvbclnqb9SXzruUfQOtCVJoxLq+fHKcuVe/eMZMehc8mYltiWm7NvyIxmdAO0vKsE2juxruzWpAYm10tOmht7+XEXiroi9DD1obSm88lFo/7NjPZre5KGdtZlY/3p/duxHIqKlrIba466Qi6HrIvryeJDElGns/gy8GCv6cixIrrbZ1MTQx2B7xIdEJMNs4Uv+bT1FD2Cm6D2+KXoQs4Sn6Buzm+gpo+gTQGJ3AP3gTA+Coh/KKOHxu3UQquf/rVP0YK6Kjufx8OUKfKECQuEZe/hs+X01ir6AK8JDZPsjwxAQPltnPAmkV3T5XdTUk7Z4IfR8PZoEUhIe9dqAzOgUPD3fACueFtgTlqLfGBGuJLKFp/cbo0WuFVxIXcjJT2TzQm5TrMitoo+4ZSPBaJFbBddcWZwhgVyd5eSGjJrl5CZwlh8IZ/lhcJYfCGf54VD0B8P76IPgitmB8CGHw+CnYAfCz7sPhKIfCE/vh8KHHA6Et2wHw8UZQubw9vYfXBQKZ4yC86UAAAAASUVORK5CYII=)
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Vernonia amygdalina
|
2
|
Vernomygdin
|
Sesquiterpene
lactones
|
![](data:image/png;base64,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)
|
Vernonia amygdalina
|
3
|
11, 13-dihydrovernodalin
|
Sesquiterpene
lactones
|
![](data:image/png;base64,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)
|
Vernonia amygdalina
|
4
|
Chicoric acid
|
Phenolic acids
|
![](data:image/png;base64,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)
|
Occinum gratissimum
|
5
|
Rosmarinic acid
|
Phenolic acids
|
![](data:image/png;base64,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)
|
Occinum gratissimum
|
6
|
Luteolin
|
Flavonoids
|
![](data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAKQAAABsCAYAAADkDhmYAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAASBSURBVHhe7d2BcdQwEIXhK4AGaIAGaIAKqIAO6IAWaIEaaIIuaCb4JYjRCK1OsiV5bf3fjCZ3PllrWw/ncsTxAwAAoK9PW/u1tZe/7fvW3m0tpuUf3x7+83VrP98e7lJTF4tRKBSGz6/PHo/3W/uxNQUl1juQtXWxmN9bC6EIdJZKl/cOZG1dLOTD1hS03LfJb1vTGSvoGciWuliIAqZg5KRhU79c2xPIlrpYCGdIuGO9ZzvjPaTwHnJx/JQNd2o+D+wdSKmpCwB+6Ew1U1xvdm1cAIGEKwQSrhBIuEIg4QqBhCsEEq4QSLhCIOEKgYQrBBJuKBChzRLXnFkXjuXCMCMgufFH14RjNaGr6dPq2ZgjasI5a8Jbl7doDVprf1xQzSRbfWrWzdm7XnBkXTi1JxTWOi1jtda0tNSEYz0m0lq/NHaPujmjxsUEPSeuFIR4ealfT7PqoIORk2WNPbJmyRk10WjGJOUCSCCRFU/S6AmbWSs4oyYOOGvCCCSyCCRcIZBwhUDCFQIJVwgkXCGQcIVAwhUCCVcIJFwhkHCFQMIVAglXZkySaqR1zggHgbyA0ZNkjb9KIFXzjLqXFA7UiINmjRkvt/r0dkZNievMqnlJ1qRYy1vsGftozZJSzVF1rbFH1ryk2gOy58CV1hlVs6R2vLNq9qx7OXsPQO06Vr89dfesEzujppTWt147WnMq3YZNd0KN6V5/ugNqoHv+6bn66t5/uRtR9jjQpQOae620Tq10/Wf72qtm6xi161j9atc/nTaydL/AcNte3fNP/TRBujuqngc9d7TmwNX0aRHGqtnXnmr2o6ZPjrXe3vGm0caVAqmveh7TWSS3Xk/WQRt5MD3tq5b12FdrnF7jd6eNsgIZJkNfUzprjDpzBPFBix+P4G1fe7PGHFHrEG2QbserAIamb1P6qqBaGxyfRUebcdBW2FeNnRt/xvGtpo35sjVNSGg6G3gK5AwrBDJQjbjOjJrVtDGajFg8AXpdNzVP6ayqIN+Jh309Iyiz6lTRxpQCqceakPi9lSZH693tTvse9jUOx6ygzKpTRRtTCqToczn10zK9v1RLP7u8i7P3NQ7HrKDMqlNFYUz/9esMkU6C+qjvXYMYO3Nflw8kfCGQcIVAwhUCCVcIJFwhkHCFQMIVAglXCCRcIZBwZVYgzwg+Lmh0ODR+WoNAwpQLTA/WuGH5iJq4kZ4BscZKlxNMFB0NiLX+s3GP1MQCngUoZfVvGaelLxb1LCClEO0NV2lMoDkgvQIVj6HrjnSpRxhbFwXGv+StZc+uQsDNhDBYnr2+l8KoccOfmNHVBfqTMwpooNcJ5KLS4KXPe9M1RunfO9LZMV5OIDE8iKJrjFQjvQZLdHGczpRCIPFqdCAVMqtGHDj1yTUCuRhN+kicIdFkdCAl9x5SeA+J/8wIJD9lo9qMQAqfQ6LKrEACVQgkXCGQcIVAwhUCCVcIJAAAbvEhNdzgv/HgCr8sCzf4VTC4opApbDlx4NQn1wgkuuIMCXf4ZVm4wk/ZcIfPIQHghh6PP5Eilr8Dp83UAAAAAElFTkSuQmCC)
|
Occinum gratissimum
|
7
|
Neoandrographolide
|
Diterpenoid lactone
|
![](data:image/png;base64,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)
|
Vernonia amygdalina
|
8
|
Vernomenin
|
Sesquiterpene
|
![](data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAIwAAABSCAYAAACG9DxhAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAATuSURBVHhe7ZyLcdUwEEVTAA3QAA3QABVQAR3QAS3QAjXQBF3QTPDNy2Y2i/5afWzfM6OJImt35dUd2ZY974kQQgghu/LpKL+O8vxa/hzl61EEtH1/VN+B9s+PKrkLEIsI4gMaDr4cBW0/Xv6jYIgCq8nPR/UdEAIEoQVloWBuBlaU1KSLUCiYTcEECLo+CllFYpP++ygimFihYBaCCRB0fRQfj5KadBzjCrMxWiS6PhKsIryHOSkrBMOnpBOjRaLrKTBhuLRY0C4CyMF9mJOiRaLrKe42maV5uQUUTBqKxbBSMOivy27YMe04xunoJJQmBP3wlANx6IJ2Cubi6CSUJgT9/h4Fj8e6oL1VMC302qewPkfEOCU6EaVJQT+vexjYlMa1iG2rfQrtc4T/09KSGPS7smCsP2//p0YnozQx6HdVwVhfnr7dQJJRSje9PNEJKU0O7lf0JpuAdmzI1YCYrZMitq32IawvT9/dIOkYEHY55aYRTx8zhaMTsiI5iNkaV2y9xm39ePl1QcSil3AIRcQzC52U2gShvy4teNi22lu0Hy+fbuCxNHQfIK//8TJuBr1Jgo2UFlrtQG9sjfXh4dON3DchMTGNQCemJUmwkdJCqx3oja3RPjz8uQKhYFAxweCSNFMwutTiZdtr34O17/XnDu5VMKjYZQfHvj2qw0CMUGJqkyV+auxi/Wv9gBYbjbXt8TUUPA2Fbm4hFAxaf2/SmxRNia+aeNK3pn+OUl+gpm8Ia9/rbxj6iQgrDS5P+NoMAw7tcQAc6zmhWtuSeKVjKu0n1PhtRWKIjx5fU4BosKJANCgQjF1ZQuiTLKG2vyVln/OdO56jxH8N4s/6rfWzLfbENLmTTNm2EPIVizEidixODrHN9S3xdRpiJxxqj/X1IBZPGBkbpGJrZBy1Y6ntvz3eCWpFx7J/RxOKDaRdt9XSY7stoaSsOlEZy4r4OrZn/BXnMgWdqJUnecXYK89pODg5Csafy4tmFRTMCaFgxnBZ0VAw4/CMgS8S8WIZZdYnLEEomHF4xcD7Q/jCjj4Eg1dD+JSl9rNWFyiYsfTGgUggDvv5LUQUah8OBTMexKotAuqhl8ryqUvshfMw9OBmc6fYLfHk93BiH8zh0jTrg7k3KJjxtMbKfWFJwUxkVmzEaSkC6ltdklaiEzObWbF742AF2eqmdyVXF4xXjC0eq3fYCLqyYLz9L52vXTaCKJgTsNNG0FUFcxmxAJzMLnfdVxTMpcSy00YQxrFaMN7xV57PEHbZCJLEyqTNTLSNt2IMpwKJWXVJshNj6/p/b0r8S59UP6zSqV8ivxxYQWbf9MYmobSth1hskIoldrqPXNKRQ8kTHnHRhoeJy1LyWG2T1ULOR+yYR2zg4QOIH+QJubPIpX72tsRUcHIQC0pqI6h18kpscn16YrfYpZBLduz+D8eQS/JK6QSUTlZpPzDCZy25B4aZv81zGlITUjtZtf2BV2yhxi736184RsFE0InW9Rq87Fp8tIJV5Lb3MB70TJad+Fp67Vu47VPSDsiEz550S+04IJpb7cPsQu1EkZtDwZAqdhMMxbs5uwiGQjkJuwiGbIwWx66CoYA3wApD/rftq9hpLLcnNAmcIPIfMUFQKOQdKUFQKOSNnFAoFvIChUKKoVBIMSFBUCikGAqFFMFVhRBCCCGEdPD09A9RbbO1Tb8UMAAAAABJRU5ErkJggg==)
|
Vernonia amygdalina
|
9
|
myricetin
|
Flavonoids
|
![](data:image/png;base64,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)
|
Occinum gratissimum
|
10
|
Isorhamnetin
|
Flavonoids
|
![](data:image/png;base64,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)
|
Vernonia amygdalina
|
|
|
|
|
|
Table 3
Binding energies of top ten ranked phytochemicals docked in the active sites of 3-Chymotrypsin-like proteases of coronaviruses.
S/N
|
Compounds
|
Binding energies (Kcal/mol)
|
SARS-CoV-2
|
SARS-CoV
|
MERS-CoV
|
S1
|
Lopinavir
|
-7.2
|
-8.3
|
-7.5
|
S2
|
Ritonavir
|
-7.2
|
-7.2
|
-7.1
|
1
|
Vernolide
|
-8.0
|
-7.9
|
-7.7
|
2
|
Vernomygdin
|
-7.9
|
-7.7
|
-7.2
|
3
|
11, 13-dihydrovernodalin
|
-7.8
|
-7.8
|
-7.2
|
4
|
chicoric acid
|
-7.7
|
-7.4
|
-8.9
|
5
|
Rosmarinic acid
|
-7.7
|
-7.2
|
-8.7
|
6
|
Luteolin
|
-7.7
|
-7.7
|
-8.3
|
7
|
Neoandrographolide
|
-7.7
|
-8.3
|
-8.1
|
8
|
Vernomenin
|
-7.7
|
-6.9
|
-6.7
|
9
|
Myricetin
|
7.7
|
-7.5
|
-8.0
|
10
|
Isorhamnetin
|
-7.6
|
-8.0
|
-8.4
|
While the top three ranked phytochemicals SARS-CoV-2 3CLpro were found to be vernolide, vernomygdin and 11, 13-dihydrovernodalin (-8.0, -7.9 and − 7.8 kcal/mol respectively); neoandrographolide, isorhamnetin and vernolide (-8.3, -8.0 and − 7.8 kcal/mol respectively) were topmost against SARS-CoV 3CLpro; and chicoric acid, rosmarinic acid and isorhamnetin (-8.9, -8.7 and − 8.4 kcal/mol respectively) against MERS-CoV (Table 3). It was observed that the top three ranked phytochemicals for SARS-CoV-2 and SAR CoV 3CLpros were isolated from Vernonia amygdalina while those for MERS-CoV 3CLpro were from Occinum gratissimum.
3.2 Molecular interactions between the top docking phytochemicals and the active sites of 3CL pro of coronaviruses
A monomer of 3CLpro is made up of three domains: domain I (residues 8–101), domain II (residues 102–184), and domain III (residues 201–303) and a long loop (residues 185–200) connects domains II and III. Domains I and II comprise six-stranded antiparallel β-barrels with the substrate binding site at the intersection of the two domains. The enzymatic activity of 3CLpro resides in the catalytic dyad of Cys145 and His41 [50]. The substrate-binding pocket lies in the cleft between domains I and II, and features the catalytic dyad residues Cys145 and His41. The substrate-binding pocket is divided into a series of subsites (including S1, S2, S4 and S1′), each accommodating a single but consecutive amino acid residue in the substrate. Ser1 in each one monomer interacts with Phe140 and Glu166 of the other monomer to stabilize the S1 subsite, a structural feature that is essential for catalysis [51]. The current study revealed that, the reference drugs and the top-docking phytocompounds are stabilized by numerous non-covalent interactions in the active regions of the target protein of the coronaviruses as shown in Table 4.
Table 4
Interacting amino acid residues of the 3-Chymotrypsin-like proteases of Coronaviruses with the top phytochemicals of Vernonia amygdalina and Occinum gratissimum
Compounds
|
Coronavirus
|
Residues involved in hydrogen bonding (bond distance, Å)
|
Residues involved in hydrophobic interactions
|
Residues involved in others interactions
|
Lopinavir (S1)
|
SARS-Cov-2
|
GLU166 (2.97) ASN142 (2.97) PRO168 (2.97) SER144(2.97)
|
MET49 HIS41 LEU27
|
CYS145
|
Ritonavir (S2)
|
SER46(2.46) THR26(3.24)
|
MET49 MET165
|
GLU166
|
Vernolide
|
GLY143 (2.00) MET165 (3.63) HIS41 (2.25)
|
CYS145
|
|
Vernomygdin
|
GLU166 (2.97) HIS163 (2.97) ASN142 (2.97) GLY143(2.97) MET165 (2.97)
|
CYS145 LEU27 MET4
|
|
11, 13-dihydrovernodalin
|
CYS145 (2.74) ASN142 (2.25)
|
MET165 HIS41 LEU27
|
|
Lopinavir (S1)
|
SARS-CoV
|
CYS145 (2.49) THR25 (2.74) GLU166 (2.10, 2.08)
|
MET49
|
HIS41
|
Ritonavir (S2)
|
THR24(2.31) HR25 (2.47) THR26(2.92) ASN142 (3.12)
|
CYS44 CYS145 MET49
|
|
Neoandrographolide
|
ASP48 (2.92) GLU166 (3.13, 3.37) GLU47 (2.47)
|
HIS41 CYS145 CYS44 MET165 MET49
|
HIS163
|
Isorhamnetin
|
HIS41 (2.47) CYS145 (2.47) MET165 (2.47) THR25(2.74) MET49 (2.47) THR24(2.74)
|
MET49
|
GLU47
|
Vernolide
|
ALA46 (2.47) THR26(2.74) GLY26 (2.74)
|
HIS41 CYS145
|
|
Lopinavir
|
MERS-CoV
|
GLN169(2.81) GLY167 (2.66)
|
HIS41 CYS145 CYS44 MET25 LEU49 ALA46
|
|
Ritonavir
|
CYS145 (3.31) CYS148 (3.67, 2.75) GLY167 (2.92, 3.03) GLY192 (2.06) ASN122 (2.34)
|
LEU49 ALA46 GLN169
|
HIS41
|
chicoric acid
|
CYS145 (3.68) GLY146 (2.36) HIS166 (2.90, 1.98) SER147 (2.48) LEU144 (2.32) THR193 (1.92) LYS191 (1.99) GLU169 (2.70)
|
LEU49
|
|
Rosmarinic acid
|
HIS41 (3.04) LEU49 (2.92) TRY54 (2.68) THR193 (2.72)
|
GLY192 MET168
|
|
Isorhamnetin
|
GLU169 (2.70) HIS41 (3.06) LEU144 (2.70)
|
MET168 CYS145
|
|
Lopinavir and ritonavir, the antiretroviral protease inhibitors which were originally developed for use against HIV and later recommended for the treatment of SARS and MERS [52], were used as reference drugs. The interactions of lopinavir were majorly through hydrogen bonds and hydrophobic interactions, with few electrostatic interactions. The 4-hydroxyl and acetyl group of lopinavir interacted via hydrogen bond to GLU166 and SER144 of the domain I and II of 3CLpro of SARS-CoV-2 while the 3-methyl and 1-phenyl moiety interacted via a carbon hydrogen interaction. The 1-phenyl and the methyl moieties of the 2,6-dimethylphenoxy interacted via hydrophobic interactions with the catalytic dyad (Cys145 and His41) residues of 3CLpro of SARS-CoV-2 (Fig. 1). For 3CLpro of SARS-CoV, the 1-amino group of 2-oxo-1,3-diazinan-1-yl, 4-hydroxyl and acetyl groups of lopinavir interacted via H-bond with GLU166, THR25 and CYS145 in the same domain as SARS-CoV-2 while the 3-methyl and 1-phenyl groups formed an alkyl and pi-sulfur interaction with MET49 and CYS145 respectively (Figure S1). In the case of MERS-CoV, two hydrogen bonds were observed between GLU169 and GLN167 and the carbonyl group and amino group of the butanamide moiety of lopinavir respectively (Figure S2), while hydrophobic interactions were formed by the phenyl rings. In the same vein, ritonavir having the same binding affinities as lopinavir interacted in a different manner with 3CLpro of the coronaviruses.
The 15-hydroxy, 7-oxatetracyclo moiety and the carbonyl group of methylprop-2-enoate of vernolide interacted via H-bond with HIS41, GLY143 and MET165 of 3CLpro of SARS-CoV-2, while the heptadec-9-en-3-yl ring formed an alkyl interaction with CYS145 (Table 4). The hydrogen bonds observed between vernomygdin and HIS163, GLU166, and GLY143 of 3CLpro of SARS-CoV-2 were contributed by dihydrofuran-2 (3H)-one and the carbonyl group of methylpropanoate. The heptadec-9-en-3-yl ring and the alkyl group of methylpropanoate moiety were responsible for the alkyl interactions with which amino acids and 3CLpro of SARS-CoV-2. The hydroxyl group of hydroxymethyl-prop-2-enoate of 11, 13-dihydrovernodalin contributed the only hydrogen bonds with CYS145 of 3CLpro of SARS-CoV-2. Several alkyl and pi-alkyl interactions were formed by the rings and methyl group of the furan ring of 11, 13-dihydrovernodalin and 3CLpro of SARS-CoV-2. Vernolide, vernomygdin and 11, 13-dihydrovernodalin, the best docked phytochemicals in the SARS-CoV-2 3CLpro were observed to interact with the S1 subsite residues such as HIS41, ASN142, GLY143, SER144 and the GLU166 residue of β11. Interactions with the S1 and β11 residues have been reported for some other inhibitors of SARS-CoV-2 replication [6, 51], suggesting that these three phytochemicals may effectively inhibit the proliferation of the virus. Interactions of the compounds at the S2 subsite were predominantly hydrophobic except for vernomygdin that formed a hydrogen bond with HIS163 and important residue in the hydrophobic pack that have been implicated in its catalytic activity [6] (Table 4). The binding of the top three ranked compounds docked in 3CLpro of the coronaviruses revealed that all the phytochemicals of V. amygdalina and isorhamnetin interacted with both amino acids of the catalytic dyad, indicating that they may be more effective inhibitors of the enzyme. The stability of the complexes formed stemmed from the vast number of interactions with some important active site residues HIS41, MET49, MET165, THR25, LEU27, ASP48, LEU50, LEU141, CYS145, HIS164, LEU167, PRO168, AEP187, and ALA191 which have been reported to be significant for the binding of the inhibitors with 3CLpro [53].
SARS-CoV 3CLpro had the highest binding affinity for neoandrographolide, a diterpene lactone obtained from V. amygdalina. The 2H-Furan-5-one ring formed two hydrogen bonds to ASP48 and GLU47. An alkyl interaction was formed by the methyl group at the oxan-2-yl-oxymethyl junction with CYS145 while the several pi-alkyl interactions were majorly formed by the 1H-naphthalen-1-yl and 2H-Furan-5-one ring (Figure S1). Isorhamnetin, an O-methylated flavon-ol obtained from Vernonia amygdalina interacted via conventional H-bonds with GLU166, GLY143 and THR45. A carbon hydrogen interaction was observed with CYS145 and THR24, while pi-cation, pi-sulfur and pi-alky were observed between the rings and HIS45, MET49 and CYS145 respectively. The carbonyl group of methylprop-2-enoate moiety and 15-hydroxyl group of vernolide formed a conventional hydrogen bond with GLY143 and THR25 of SARS-CoV 3CLpro. Pi-alkyl and alkyl interactions of the heptadec-9-en-3-yl with HIS41 and CYS145 were also observed.
Chicoric acid, a constituent of Occinum gratissimum, was the best docked phytochemical to MERS-CoV 3CLpro. The hydroxyl and carbonyl groups on prop-2-enoyl[oxy]butanedioic moiety of chicoric acid interacted via several H-bonds with the residues at the active site. The hydroxyl and carbonyl groups on prop-2-enoyl[oxy]propanoic moiety of rosmarinic acid contributed the 3 hydrogen bonds to TYR54, LEU49 and HIS41 (Figure S2), while the first 3,4-dihydroxyphenyl moiety formed the hydrophobic interactions. HIS41 formed both hydrogen bond and pi-pi T-shaped interaction with the carbonyl group on the chromen-4-one moiety of isorhamnetin. The 4-hydroxy-3-methoxyphenyl moiety of isorhamnetin formed carbon hydrogen and pi-alkyl interactions with CYS145 (Figure S2).
3.3 Optimization of docking interactions of phytocompounds with SARS-CoV-2 3CL Pro conformations
An in-depth docking simulation of the phytocompounds was performed to validate their docking and interactions with the target protein. Figure 2 shows the average binding affinities of the best ten phytocompounds along with the reference inhibitors (positive controls) against the five different representative conformations gotten from the clustering analysis of the SARS-CoV-2 3CLpro MDS trajectories (see Figure S3). The means and the standard errors of the mean of the 5 binding energies for each representative conformation of SARS-CoV-2 3CLpro were calculated for each phytochemicals and reference inhibitors. As reflected from the binding energy values, the ten phytochemicals are able to bind effectively to the SARS-CoV-2 3CLpro different conformations, just like the positive controls. The binding energy values ranged from − 6.1 Kal/mol (rosmarinic acid) down to -8.1 Kcal/mol (neoandrographolide and chicoric acid). As reflected from Fig. 2, vernolide, neoandrographolide, myricetin, chicoric acid, luteolin, and Isorhamnetin (green columns) are the compounds with best the binding affinities to SARS-CoV-2 3CLpro. To further analyze the data, we examined the docking complexes of these best compounds using the PLIP analysis web server. We examined the docking complexes of these best compounds using the PLIP analysis webserver to further analyze the data.
From the docking results, five complexes for each phytochemical were generated. The best representative complex for each phytochemical was selected based on the binding affinity for further analysis using the PLIP webserver. The details of the interactions established upon docking of the reference inhibitors and the best ten phytochemicals against SARS-CoV-2 3CLpro are presented in Table 5. The most reported types of interactions are hydrogen bonding and few hydrophobic contacts in some complexes. At least three hydrogen bond, and up to seven were reported in the docking complexes between the compounds and SARS-CoV-2 3CLpro. The most-reported residues from the 3CLpro that interacted with the ligands (represented in bold in Table 5) are ASN142, GLY143, SER144, CYS145, and GLU166, and these formed 6, 9, 14, 7, and 5 interactions with the ligands, respectively. CYS145 is one of the 3CLpro active site dyads (HIS41 and CYS145), and it was reported in all the ligands except myricetin and luteolin.
Table 5
The interactions of the top 10 ranked phytochemicals of Vernonia amygdalina and Occinum gratissimum and positive control (Ritonavir and Lopinavir) for the best representative conformation from the cluster analysis of SARS-CoV-2 3CLpro molecular dynamics simulation (MDS) trajectories
Compound
|
Binding energies
(kcal/mol)
|
H-bonding
|
Hydrophobic interactions
|
Number
|
Residues from SARS-CoV-2 Mpro
|
Number
|
Residues from SARS-CoV-2 Mpro
|
Ritonavir
|
-6.4
|
6
|
ASN142(2), GLY143, SER144, CYS145, and GLU166
|
1
|
MET165
|
Lopinavir
|
-6.3
|
5
|
ASN142, GLY143, ASP178(2), and GLN189
|
1
|
THR25
|
Vernolide
|
-7.5
|
3
|
GLY143, SER144, and CYS145
|
1
|
MET165
|
Vernomygdin
|
-6.9
|
5
|
ASN142, GLY143, SER144, CYS145, and GLN189
|
2
|
MET165, and GLU166
|
11, 13-dihydrovernodalin
|
-6.6
|
6
|
ASN28(2), GLY143, SER144, CYS145, and GLU166
|
3
|
LEU27(2), and MET165
|
Neoandrographolide
|
-7.7
|
7
|
THR45, SER46, LEU50, ASN142, GLY143, SER144, and CYS145
|
1
|
THR25
|
Vernomenin
|
-6.4
|
3
|
GLY143, SER144, and CYS145
|
2
|
THR25, and LEU27
|
Myricetin
|
-7.1
|
7
|
LEU141, ASN142, GLY143, SER144 (3), and GLU166
|
0
|
|
Chicoric acid
|
-7.3
|
6
|
LEU141, ASN142, GLY143, SER144 (2), and CYS145
|
1
|
GLN189
|
Luteolin
|
-7.2
|
4
|
SER144, GLU166(2), and GLN189
|
0
|
|
Rosmarinic acid
|
-6.8
|
7
|
THR26(2), PHE140, LEU141, GLY143, SER144, and CYS145
|
0
|
|
Isorhamnetin
|
-7.4
|
6
|
ASN142, GLY143, SER144 (3), and CYS145
|
0
|
|
Residues in bold represent the most reported residues that interacted with the compounds. |
So far, two terpenoid structures viz: vernolide and neoandrographolide with strong interactions with the active region of SARS-CoV-2 3CLpro have been identified (Fig. 2, Table 5 and Fig. 3). The surface views of these structures in the substrate binding pocket of SARS-CoV-2 3CLPro are shown in Fig. 4.
Binding interactions of neoandrographolide at the enzyme catalytic site is stabilized by several H bonds between its 2H-Furan-5-one ring and key residues (ASN142, GLY143, SER144, CYS145) of catalytic pocket of the enzyme, which led this ring to be sandwiched between CYS145 and ASN142 (Fig. 3). Furthermore, neoandrographolide structure inserts into the bulky hydrophobic S1/S2 subsites (composed of the side chains of HIS41, MET49, HIS41, ASN142, GLY143, SER144, and MET165) (Fig. 3 and Fig. 4b). Consequently, neoandrographolide was accommodated in the substrate-binding pocket and interacted with the catalytic residues, the oxyanion loop (residues 138–145), and the S1/S2 subsites, which are the key elements for the recognition of substrates. Interactions with the S1 have been reported for some other inhibitors of SARS-CoV-2 replication [6, 51] suggesting that this structure may effectively inhibit the proliferation of the virus. With the aid of an array of direct and indirect hydrogen bonds with ASN142/GLY143/SER144/CYS145, neoandrographolide may fix the conformation of the flexible oxyanion loop, which served to stabilize the tetrahedral transition state of the proteolytic reaction. This binding mode of neoandrographolide is similar in many respect to that of baicalein, the first natural noncovalent, nonpeptidomimetic inhibitor of SARS-CoV-2 3CLpro derived from Shuanghuanglian [54]. Vernolide, another terpenoid structure (sesquiterpene lactone) isolated from Vernonia amygdalina is another potential non-covalent inhibitor of SARS-CoV-2 3CLPro inhibitor. Its interactions with the active site of this enzyme mimic the non-covalent interactions of carmofur, a potent covalent inhibitor of this enzyme which also establishes non-covalent interactions with its target [55]. The carbonyl group of methylprop-2-enoate moiety of vernolide occupies the oxyanion hole and forms hydrogen bonds with the backbone amides of Gly143, and Cys145 (Fig. 3 and Fig. 4a), mimicking the tetrahedral oxyanion intermediate formed during protease cleavage. A side chain of vernolide inserts into the bulky hydrophobic S2 subsite (composed of the side chains of HIS41 and MET165) (Fig. 3 and Fig. 4a). Therefore, these terpenoid structures alongside other phytocompounds from the source plants may be suggested as inhibitors of SARS-CoV-2 3CLpro.
3.7 In Silico Drug-likeness and Pharmacokinetic properties of vernolide, Neoandrographolide, and other topmost phytocompounds
The top 6 phytocompounds (Neoandrographolide, Vernolide, Isorhamnetin, Chicoric acid, Luteolin, and Myricetin) from the docking analysis to the representative conformation gotten from the clustered MDS trajectories were subjected to the predictive drug-likeness and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) filtering analyses. The result of the analyses for the six-top phytocompounds is represented in the Table 6. ability to pass the high human intestinal absorption, low acute oral toxicity with a good bioavailability score (Table 5).
Table 6
In silico Physicochemical and ADMETa parameters of the top-binding phytochemicals of Vernonia amygdalina and Occinum gratissimum with 3-Chymotrypsin-like protease of SARS-CoV-2
a) Physicochemical properties
|
Vernolide
|
Neoandrographolide
|
Isorhamnetin
|
Chicoric acid
|
Luteolin
|
Myricetin
|
Molecular weight (g/mol)
|
362.37
|
480.59
|
316.26
|
474.37
|
286.23
|
318.24
|
Num. heavy atoms
|
26
|
34
|
23
|
34
|
21
|
23
|
Num. arom. Heavy atoms
|
0
|
0
|
16
|
12
|
16
|
16
|
Num. rotatable bonds
|
3
|
7
|
2
|
11
|
1
|
1
|
Num. H-bond acceptors
|
7
|
8
|
7
|
12
|
6
|
8
|
Hydrogen bond donor
|
1
|
4
|
4
|
6
|
4
|
6
|
cLogP
|
0.93
|
2.63
|
1.87
|
2.01
|
2.52
|
1.18
|
Molar Refractivity
|
89.51
|
125.27
|
82.50
|
114.00
|
76.01
|
80.06
|
TPSA (Ų)
|
94.59
|
125.68
|
120.36
|
208.12
|
111.13
|
151.59
|
|
Drug-likeness
|
Lipinski
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
Yes
|
Veber
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Ghose
|
Yes
|
No
|
Yes
|
No
|
Yes
|
No
|
Egan
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
No
|
Muegge
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
No
|
Bioavailability Score
|
0.55
|
0.55
|
0.55
|
0.11
|
0.55
|
0.55
|
|
Absorption (Probability)
|
(b) Admet SAR
|
|
|
|
|
HIA
|
HIA+ (0.58)
|
HIA- (0.127)
|
HIA- (0.498)
|
HIA+ (0.883)
|
HIA+ (0.9650)
|
HIA- (0.437)
|
Caco-2 Permeability Cm/s
|
Caco2+ (-5.096)
|
Caco2+ (-5.84)
|
Caco2+ (-5.217)
|
Caco2+ (-6.709)
|
Caco2+ (-5.12)
|
Caco2+ (-6.63)
|
P-glycoprotein Substrate
|
Neg. (0.484)
|
Pos. (0. 778)
|
Neg. (0.015)
|
Neg. (0.051)
|
Neg. (0.038)
|
Neg. (0.208)
|
P-glycoprotein Inhibitor
|
Neg. (0.027)
|
Neg. (0.007)
|
Pos. (0.538)
|
Neg. (0.193)
|
Neg. (0.366)
|
Neg. (0.064)
|
|
Distribution (Probability)
|
Blood-Brain Barrier
|
BBB+ (0.4.39)
|
BBB- (0.476)
|
BBB- (0.34)
|
BBB+ (0.552)
|
BBB-(0.464)
|
BBB- (0.4.27)
|
PPB %
|
65.501
|
72.039
|
90.707
|
76.782
|
91.796
|
76.595
|
VD L/kg
|
-0.147
|
-0.452
|
-0.932
|
-1.406
|
-1.406
|
-1.39
|
|
Metabolism (Probability)
|
CYP450 1A2 Inhibitor
|
Neg. (0.069)
|
Neg. (0.028)
|
Pos. (0.941)
|
Neg. (0.239)
|
Neg. (0.069)
|
Neg. (0.133)
|
CYP450 1A2 Substrate
|
Neg. (0.33)
|
Neg. (0.258)
|
Neg. (0.456)
|
Neg. (0.262)
|
Pos. (0.968)
|
Pos. (0.968)
|
CYP450 3A4 Inhibitor
|
Neg. (0.149)
|
Neg. (0.262)
|
Pos. (0.768)
|
Neg. (0.087)
|
Neg. (0.412)
|
Neg. (0.376)
|
CYP450 3A4 Substrate
|
Neg. (0.562)
|
Neg. (0.523)
|
Neg. (0.428)
|
Neg. (0.15)
|
Pos. (0.867)
|
Neg. (0.459)
|
CYP4502C9 Inhibitor
|
Neg. (0.116)
|
Neg. (0.144)
|
Neg. (0.183)
|
Neg. (0.071)
|
Neg. (0.328)
|
Pos. (0.656)
|
CYP450 2C9 Substrate
|
Neg. (0.313)
|
Neg. (0.408)
|
Pos. (0.772)
|
Pos. (0.504)
|
Neg. (0.0496)
|
Pos. (0.557)
|
CYP4502C19 Inhibitor
|
Neg. (0.093)
|
Neg. (0.103)
|
Neg. (0.24)
|
Neg. (0.157)
|
Neg. (0.124)
|
Neg. (0.068)
|
CYP450 2C19 Substrate
|
Neg. (0.474)
|
Neg. (0.462)
|
Pos. (0.54)
|
Neg. (0.334)
|
Pos. (0.542)
|
Neg. (0.345)
|
CYP4502D6 Inhibitor
|
Neg. (0.296)
|
Neg. (0.329)
|
Neg. (0.468)
|
Neg. (0.248)
|
Neg. (0.463)
|
Neg. (0.318)
|
CYP450 2D6 Substrate
|
Neg. (0.267)
|
Neg. (0.274)
|
Neg. (0.41)
|
Neg. (0.415)
|
Neg. (0.401)
|
Neg. (0.18)
|
|
Elimination
|
T 1/2 (Half Life Time)
|
0.883 h
|
1.53 h
|
0.658 h
|
1.79 h
|
0.745 h
|
1.915 h
|
CL (Clearance Rate)
mL/min/kg
|
1.914
|
1.032
|
1.951
|
0.823
|
1.919
|
1.709
|
|
Toxicity
|
hERG Blockers
|
Ng. (0.256)
|
Neg. (0.474)
|
Neg. (0.301)
|
Neg. (0.578)
|
Neg. (0.436
|
Neg. (0.353)
|
H-HT
|
Neg. (0.444)
|
Pos. (0.584)
|
Pos. (0.654)
|
Neg. (0.348)
|
Pos. (0.592)
|
Neg. (0.332)
|
AMES
|
Neg. (0.411)
|
Neg. (0.224)
|
Neg. (0.044)
|
Neg. (0.224)
|
Pos (0.74)
|
Neg. (0.074)
|
SkinSen
|
Neg (0.340
|
Neg (0.256)
|
Neg (0.186)
|
Neg (0.414)
|
Neg (0.278)
|
Neg. (0.278)
|
LD50 (LD50 of acute toxicity)
|
3.211 -log mol/kg
(222.927 mg/kg)
|
3.448-log mol/kg
(171.31 mg/kg)
|
2.71-logmol/kg
(604.02mg/kg)
|
2.38-logmol/kg
(1945.92mg/kg)
|
2.58 -log mol/kg
(737.444 mg/kg)
|
2.69 -log mol/kg
(648.262 mg/kg)
|
DILI
|
Neg. 0.424
|
Neg. (0.196)
|
Pos. 0.904
|
Pos. 0.84
|
Pos. 0.9
|
Pos. 0.9
|
|
Pharmacokinetics
|
GI absorption
|
High
|
High
|
High
|
High
|
High
|
High
|
Log Kp (skin permeation) cm/s
|
-7.85
|
-7.36
|
-6.93
|
-7.77
|
-6.25
|
-7.40
|
aADMET: Absorption, distribution, metabolism, elimination, and toxicity; GI: Gastro-intestinal; BBB: Blood Brain Barrier; P-gp: permeability glycoprotein; CYP: cytochrome P450; hERG: human Ether-à-go-go-Related Gene; HIA : Human Intestinal Absorption; H-HT: Human Hepatotoxicity AMES: Ames Mutagenicity; DILI: Drug Induced Liver Injury; VD: Volume Distribution; PPB: Plasma Protein Binding |
From these six, two phytocompounds ( vernolide, neoandrographolide, isorhamnetin and luteolin), fulfilled the requirement for at least four from the five physicochemical analysis (Lipinski, Veber, Ghose, Egan and Muegge) analysis, thereby suggesting favourable physicochemical/druggable properties (Table 6) [47, 56]. Vernolide, expressed positive and high probability of human intestinal absorption and non-substrate to the permeability-glycoprotein (P-gp). It is thereby suggested that the phytocompounds will be absorbed into the blood stream subverting the capability of P-gp to pumps them back into the intestinal lumen, bile ducts, urine-conducting ducts and capillaries respectively [57]. Blood brain barrier (BBB) penetration, predicts the blood brain barrier penetration of a molecule. Vernolide displayed properties that indicated their ability to cross the BBB. SARS-CoV-2 has been reported to infect the brain, thus indicating its ability to cross the blood brain barrier (BBB) [58], compounds that can cross the BBB will be beneficail in the overal all viral clearance. compounds that can cross the BBB will be beneficail in the overall viral clearance. The estimated half-life time (less than 2 hours) and clearance ratefall within the moderate range. Vernolide, neoandrographolide, isorhamnetin presented a tolerable LD50 between (51~500 mg/kg), Among the descriptors for the in silico toxicities analysis, hERG channel plays a vital role in the repolarization and termination stages of action potential in cardiac cells [59]. Compounds that block the hERG channel have the potential to cause cardiotoxicity [60]. All the six phytocompounds did not exhibit the potential of being hERG channel blockers, suggesting that they may not cause hERG channel-related cardiotoxicity. The three compounds did not exhibited mutagenicity in silico, thereby they may notcause genetic mutations, which do initiate the pathophysiology of other diseases, such as cancer. The impact of the compounds on phase I drug metabolism in the liver was also analysed using the various cytochrome P450 descriptors. Vernolide, neoandrographolide did not display inhibitory potential for the various cytochrome P450, thus may not adversely affect phase I drug metabolism in the liver. Hence, vernolide, neoandrographolide seem to demonstrate high probability of absorption, subcellular distribution, and low toxicity. The ADME/tox analysis indicated high aqueous solubility.
3.8 Molecular Dynamic Simulations and binding free energy calculation for the best two complexes.
MDS for the best two complexes were performed for 25 ns using NAMD software, and then the MM-GBSA was done using Amber tools. In Table 7, the residual contribution for the binding of 3CLpro and the best two compounds (Neoandrographolide and Vernolide) are listed with the bold residues for the highest contributed residues in the binding (bold). The active site dyads (H41 and C145) are shown underlined in the table as well. For the 3CLpro- Neoandrographolide complex, C44 is the main contributor for the binding (-1.20 kcal/mol), while for the 3CLpro-Vernolide complex, H41, C145, and M165 are the main contributors (-1.18, -1.00, and − 1.24 kcal/mol, respectively). The contribution of the active site dyads (H41 and C145) of the 3CLpro in the binding of Vernolide to the protein is evident from Table 7 (-2.18 kcal/mol). In comparison, a lower contribution of these two residues was reported in the case of the 3CLpro- Neoandrographolide complex (-0.36 kcal/mol).
Table 7
The MMGBSA calculations for the best two complexes after 25 ns MDS. Bold residues are that have binding energy greater than or equal to 0.5 kcal/mol.
complex
|
3CLpro-Neoandrographolide complex
|
3CLpro-vernolide complex
|
|
Residue
|
Binding energy (kcal/mol)
|
Residue
|
Binding energy (kcal/mol)
|
C44
|
-1.20
|
M165
|
-1.24
|
P52
|
-0.72
|
H41
|
-1.18
|
Q189
|
-0.69
|
C145
|
-1.00
|
L50
|
-0.66
|
H164
|
-0.91
|
N51
|
-0.50
|
L27
|
-0.88
|
R188
|
-0.42
|
Q189
|
-0.36
|
S46
|
-0.37
|
T25
|
-0.26
|
C145
|
-0.26
|
C44
|
-0.20
|
R40
|
-0.25
|
V42
|
-0.19
|
T45
|
-0.21
|
V186
|
-0.19
|
T25
|
-0.15
|
P39
|
-0.18
|
G143
|
-0.14
|
R40
|
-0.18
|
V186
|
-0.11
|
G143
|
-0.09
|
H41
|
-0.10
|
G146
|
-0.08
|
V42
|
-0.09
|
F181
|
-0.07
|
H163
|
-0.09
|
D187
|
-0.07
|
S144
|
-0.07
|
R188
|
-0.06
|
M165
|
-0.07
|
|
|
C38
|
-0.06
|
|
|
N142
|
-0.06
|
|
|
tOTAL BINDING ENERGY
|
-6.50 kcal/mol
|
-8.51 kcal/mol
|
Figure 5 supports the previous results when the Root Mean Square Fluctuations (RMSF) in Å was plotted for the apo-3CLpro (blue line) and the two complexes (3CLpro- Neoandrographolide (red line) and 3CLpro-Vernolide (green line)). Two regions of the RMSF plots have higher fluctuations (greater than 2Å) in addition to the N and C termini, the S46-P52 region (red cartoon) and the T190-A193 region (yellow cartoon). As shown in the RMSF at the S46-P52 region, the apo-3CLpro shows higher fluctuations than the 3CLpro- Neoandrographolide and 3CLpro-Vernolide. This region forms a loop that is important in substrate recognition since its presence near the protein's active site (blue sticks). The stabilization effect of the ligand binding to that region of the protein is due to C44 (magenta sticks) in the case of 3CLpro- Neoandrographolide, which has the most contribution in the protein-ligand binding (-1.20 kcal/mol). In comparison, H41 and C145 (blue sticks) are the most contributed residues in the binding in 3CLpro-Vernolide (-2.18 kcal/mol).