Mutations in Main Protease of SARS CoV-2 Decreased Boceprevir Affinity

Abstract

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global health emergency. The main protease (M^pro) is crucial for the life cycle of coronaviruses. Boceprevir is a potential inhibitor and drug candidate for the M^pro of SARS-CoV-2. In this study, changes in the protein structure of the M^pro due to mutations in SARS-CoV-2 and the effects of these changes on boceprevir affinity, an important potential therapeutic agent, were investigated. The mutations were analyzed with RDP4 and MegaX. A three-dimensional model of mutant M^pro was generated by ProMod3. Qualitative Model Energy Analysis, ProSA, and MolProbity tools were used for structural validation and modeling of the wild-type and mutant M^pro proteins. Topological differences of the wild-type and mutant M^pro were calculated with the i-Tasser TM-Score. Molecular docking was performed using AutoDock 4.2. Functional dynamic structure models were created with DynOmics. Seven mutations (L89F, K90R, P108S, A191V, T224A, A234V and S254F) were detected in the M^pro of SARS-CoV-2. The mutations caused a decrease in the affinity of boceprevir, a potential protease inhibitor. The boceprevir was docked to the active site of M^pro, and the binding energies were −10.34 and −9.41 kcal.mol^-1 for the wild-type and the mutant, respectively. The Debye-Waller factors calculated by elastic network model analysis were 0.58 and 0.64 Ų for the wild-type M^pro and mutant M^pro, respectively. Mutations in structures that are important drug targets for SARS-CoV-2 may render existing therapeutics ineffective in its treatment.

Keywords:
boceprevir affinity; drug validity; main protease; mutation; SARS-CoV-2

HIGHLIGHTS

• Boceprevir is a potential therapeutic that targets the main protease of SARS-CoV-2.

• Mutations change the inhibitory binding pattern in the catalytic domain of the main protease.

• Mutations of main protease of SARS-CoV-2 cause a decrease in boceprevir affinity.

HIGHLIGHTS

• Boceprevir is a potential therapeutic that targets the main protease of SARS-CoV-2.

• Mutations change the inhibitory binding pattern in the catalytic domain of the main protease.

• Mutations of main protease of SARS-CoV-2 cause a decrease in boceprevir affinity.

INTRODUCTION

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the etiological agent responsible for the global coronavirus disease 2019 (COVID-19) outbreak [¹]. SARS-CoV-2 emerged in China at the end of 2019 and spread all over the world in a short time [²]. SARS-CoV-2 killed 3.1 million people in the past sixteen months [³]. Apart from SARS-CoV-2, it is known that there are six types of coronaviruses that cause diseases in humans. Four of these (229E, OC43, NL63, and HKU1) are common and typically cause flu symptoms [⁴]. The other two strains-severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)-are of zoonotic origin and deadly pathogens [⁵]. The high prevalence, wide distribution, genomic diversity, and high recombination potential of coronaviruses reveal the fact that the new members of this virus family, which appear from time to time, may infect humans in the future [⁵,⁶].

Studies on vaccines for prophylaxis of COVID-19 have recently been successful, but a specific therapeutic drug molecule is still being developed. Generally, there are studies to reposition the drugs used for treatment of different diseases for the treatment of COVID-19. Although favipiravir, ribavirin, chloroquine, merimepodib, and remdesivir, which are used in the treatment of different diseases, are not specific therapeutic drugs for COVID-19, they are used despite their limited contribution to the healing process of the disease [⁷-⁹]. Therefore, the development of specific drugs is urgent.

SARS-CoV-2 is a positive-polarity RNA virus. The genomes of RNA viruses show a high risk of mutation. One of the important reasons affecting the spread rate and mortality rate of the disease is mutations in SARS-CoV-2 strains [¹⁰]. More than 12,000 mutations have been identified in the SARS-CoV-2 genome to date. While some of these mutations do not cause any changes in the virulence properties of the virus, some of them may have negative results in many factors ranging from virulence features to treatment sensitivity [¹¹-¹³].

The SARS-CoV-2 genome has a single-chain, 29.9 kilobase size, organization of 12 structural proteins [¹⁴,¹⁵]. The major open reading frame (ORF) 1ab encodes for two overlapping polyproteins (pp1a and pp1ab), which are cleaved into 16 nonstructural proteins by the main protease (M^pro or known as 3CL^pro) and the papain-like protease (PL^pro) [¹⁶-¹⁹]. These nonstructural proteins regulate the production of SARS-CoV-2 basic structural (spike, envelope, membrane and nucleocapsid) and accessory proteins. Therefore, M^pro is crucial for the life cycle of coronaviruses [²⁰]. M^pro cleaves pp1ab at 11 distinct sites. It is considered an important antiviral drug target because it is structurally different from human proteases [⁹,²¹]. On the other hand, PL^pro recognizes the C-terminal sequence of ubiquitin. For this reason, PL^pro inhibitors derived from the substrate have the disadvantage of also inhibiting host-cell deubiquitinases [²⁰]. SARS-CoV-2 M^pro is known to be similar to other coronaviral main proteases. In particular, the high structural similarity in the active site enables the use of previous gains for treatment in SARS-CoV-2 M^pro [²²-²⁴]. Viral proteases are promising targets for many different viruses, including hepatitis C virus (HCV) and human immunodeficiency virus. Since there are no specific antiviral drugs clinically approved to combat SARS-CoV-2, the use of clinically approved drugs for different diseases is urgently required to treat COVID-19 [²⁵]. Boceprevir is a peptidomimetic ketoamide serine protease tripeptide inhibitor used in the treatment of HCV genotype 1 [²⁶,²⁷]. Although boceprevir is not a specific drug for SARS-CoV-2, it is thought to be a potential drug candidate for SARS-CoV-2 due to its inhibition success on the protease of the HCV [²⁵,²⁸-³⁰]. Many studies have shown that boceprevir also effectively inhibits the SARS-CoV-2 M^pro [²⁵,³¹].

The urgent demand for the development of drugs effective against SARS-CoV-2 requires the use of in silico methods for rapid screening of potential therapeutic molecules. Many in silico studies have suggested potential inhibitors against proteins of SARS-CoV-2 [³²-³⁴]. In this study, changes in the protein structure of the main protease due to mutations in SARS-CoV-2 North American isolates and the effects of these changes on boceprevir affinity, an important potential therapeutic agent for SARS-CoV-2, were investigated by in silico techniques. This study aims to provide critical information on the optimization and design of stronger inhibitors against SARS-CoV-2.

MATERIALS AND METHODS

Sequence and Mutation Data

In this study, information on the nucleotide and protein sequence of 319 isolates from the North American continent belonging to the SARS-CoV-2 M^pro was obtained from the NCBI Virus database (NCBI 2020). The reference M^pro accession code is YP_009725301.1. The protein sequence information of 319 isolates was aligned with the MAFFT (v7.471) multiple sequence alignment program FFT-NS-i algorithm [³⁵-³⁷]. The scoring matrix BLOSUM 80 was chosen for the amino acid sequences [³⁸]. A value of 2.0 was used for gap opening penalty. The mutated residues were analyzed with the RDP4 and MegaX [³⁹,⁴⁰].

Homology Model of Mutant Protein

A three-dimensional model of mutant-type SARS-CoV-2 M^pro was generated by the method of homology modeling using Swiss-Model [⁴¹]. 6zru (RCSB protein data bank code) was selected as the template. Boceprevir, dimethyl sulfoxide, and water molecules were removed from the template structure. Models were built based on the target-template alignment using ProMod3 (ver3.1.1) [⁴²]. Qualitative Model Energy Analysis (QMEAN), ProSA, and MolProbity tools were used for structural validation and modeling of the wild-type and mutant M^pro proteins [⁴³-⁴⁵]. Secondary structure components (random coils, beta strands, and alpha helices) of the M^pro protein were defined with PSIPRED [⁴⁶]. Superimpose and conformational analyses of the wild-type and mutant proteins were performed with UCSF Chimera (ver1.14) and PyMOL (ver2.4.1) [⁴⁷]. Topological differences of the wild-type and mutant M^pro were calculated with the i-Tasser TM-Score and root mean square deviation (RMSD) algorithm [⁴⁸,⁴⁹].

Docking

The modeled structures of both wild-type and mutant were used as targets, and boceprevir (CID 10324367) was used as the ligand for molecular docking using AutoDock 4.2 [⁵⁰]. Kollman charges were added to the M^pro wild-type and mutant models. Gasteiger partial charges were applied to the ligands. Docking was performed with a grid dimension of 68 × 100 × 98 (−15.989, −30.532, 8.404) with a grid spacing of 0.375 Å around the binding pocket. Docking simulations were performed with the Lamarckian genetic algorithm (LGA) [⁵¹]. The main selected LGA parameters were 100 runs, 2.7 × 10⁴ generations, and 300 population size. A maximum of 2.5 × 10⁷ energy evaluations was applied for each experiment. The results were clustered according to binding energy scores using a tolerance of 2.0 Å RMSD. Docking results were visualized with Discovery SV (ver20.1, DDS Biovia).

Dynamic Network Models

Functional dynamic structure models in the protein structure before and after mutations were created with the DynOmics portal. Coarse-grained models for conformational dynamic analysis for the wild-type and mutant proteins were created with the Anisotropic Network Model and the Gaussian Network Model (GNM) [⁵²,⁵³].

Protein Stability and Ligand Affinity Analysis

Analysis of changes in protein stability was performed using DynaMut2 [⁵⁴], SDM [⁵⁵], mCSM stability [⁵⁶] and DUET [⁵⁷]. The change in boceprevir affinity before and after the mutation was evaluated using the CSM-lig program [⁵⁸].

RESULTS

Mutations of the SARS-CoV-2 M^pro caused a decrease in the affinity of boceprevir, a potential protease inhibitor for SARS-CoV-2. Seven mutations (L89F, K90R, P108S, A191V, T224A, A234V, and S254F) in the M^pro of SARS-CoV-2 were detected in the North American isolates (Table 1).

The mutation data were processed on the M^pro sequence information of SARS-CoV-2. The mutant M^pro was modeled. For the mutant M^pro model, the QMEAN score was −0.03, the Ramachandran favored score was 98.3%, the Ramachandran outlier was 0.33%, and the MolProbity score was 0.9. The Z-score of the mutant model was −7.06, and the model was found in the native protein range that was identified by X-ray crystallography (Figure 1).

Protein stability analyses data showed that the mean distance change was 3.81 Å, the total energy change was −4.22 kcal/mol, and the change in protein stability was −1.06 kcal/mol. While the residues 89,108,191,234 and 254 decreased the molecular flexibility, the residues number 90 and 224 increased the molecular flexibility. While mutations at residues 89,90,108,191,224 and 254 caused destabilization of the protein, the mutation at residue 234 contributed to protein stabilization (Table 2). Boceprevir affinities were 10.0 and 9.4 (−log₁₀ (KD / Ki)) in the wild-type and mutant proteins, respectively.

Table 1
Mutations of SARS-CoV-2 M^pro.

Mutant site	Mutant sequence ID
89 L>F	QNR99561.1, QNN95411.1, QNN95747.1, QNN96047.1, QNL12792.1, QNL12876.1, QNN95351.1, QNL36356.1, QNL36368.1, QNK39067.1, QNK39031.1, QNK39043.1, QNK39055.1, QNR99357.1, QNR99393.1, QNR99429.1, QNN95807.1, QNN95483.1, QNR99441.1, QNR99477.1, QNR99381.1, QNN96107.1, QNR99417.1, QNN95327.1, QNN96071.1, QNN96119.1, QNS00107.1, QNL12684.1, QNL13128.1, QNR54217.1 QNR99501.1, QNR99549.1, QNS00095.1, QNS00119.1, QNL12504.1, QNL12516.1, QNL10944.1, QNL12648.1, QNL12540.1, QNN95423.1, QNL12576.1, QNL12588.1, QNL12528.1
90 K>R	QNL12744.1, QNL11508.1, QNL11280.1
108 P>S	QNR99345.1, QNS00143.1, QNS00155.1, QNN95711.1, QNN95891.1
191 A>V	QNR99357.1, QNR99393.1, QNR99441.1, QNR99477.1
224 T>A	QNL36356.1, QNL36368.1, QNK39067.1, QNK39031.1, QNK39043.1, QNK39055.1
234 A>V	QNN95351.1, QNR99357.1, QNR99393.1, QNR99441.1, QNR99477.1
254 S>F	QNL10956.1, QNL11028.1, QNL11112.1

Figure 1
Z score - Model quality of the mutant SARS-CoV-2 M^pro.

Superimposition of the mutant and the wild-type structures showed that the RMSD value was 0.072 Å and the TM-Score was 0.9. Boceprevir was docked to the active site of the M^pro: the binding energies were between −10.34 and −6.14 kcal.mol^-1 and between −9.41 and −4.98 kcal.mol^-1 for the wild type and mutant type, respectively. While the wild-type M^pro interacted with boceprevir in 32 different conformations, the mutant M^pro interacted in 35 different conformations (Table 3).

The lowest binding energy and inhibitor concentration for boceprevir were −10.34 kcal.mol^-1 and 0.03 µM for the wild-type M^pro, respectively. For the conformation at this binding energy, boceprevir formed a hydrogen bond with the wild-type M^pro between the Gly143:N and Boc:O36 residues, between the His41:NE2 and Boc:O4 residues, between the Cys134:SG and Boc:N11 residues, and between Thr26:O and Boc:N37 residues. The boceprevir formed a C-H bond with the wild-type M^pro between the His164:O and Boc:C19 residues. Boceprevir formed alkyl interactions between the Met49, Met165, and Pro168 residues and pi-alkyl interactions between residues His41 and His163 (Figure 2).

Table 2
Change in SARS-CoV-2 M^pro stability.

			ΔΔG (kcal.mol-1)					ΔΔSVib (kcal.mol-1.K-1)
wild	position	mutant	RSA (%)	mCSM	SDM	DUET	ENCoM	DynaMut	ENCoM	Flex.	Stability
L	89	F	0.0	-1.851	-0.66	-1.803	0.182	-0.108	-0.228	Dec.	Destabilizing
K	90	R	47.6	-0.887	-0.21	-0.554	-0.040	0.272	0.049	Inc.	Destabilizing
P	108	S	32.4	-1.48	-0.55	-1.428	0.142	-0.585	-0.177	Dec.	Destabilizing
A	191	V	98.4	-0.327	-0.04	-0.014	0.272	0.440	-0.340	Dec.	Destabilizing
T	224	A	67.0	-0.675	0.15	-0.434	-0.646	-0.240	0.807	Inc.	Destabilizing
A	234	V	0.8	0.204	-1.03	0.384	0.421	-0.242	-0.527	Dec.	Stabilizing
S	254	F	20.8	-1.032	1.02	-0.568	0.185	0.315	-0.231	Dec.	Destabilizing

• Dec.: Decrease, Inc.: Increase, Flex: Flexibility, RSA: Residue relative solvent accessibility, SDM: Site direct mutator, ΔΔS_Vib: Δ Vibrational entropy energy between wild-type and mutant.

Table 3
Docking results ranked by energy.

Row	wild		mutant		Row	wild		mutant
	Binding energy kcal/mol	Kie µM	Binding energy kcal/mol	Kie µM		Binding energy kcal/mol	Kie µM	Binding energy kcal/mol	Kie µM
1	-10.34	0.03	-9.41	0.13	19	-7.24	4.97	-7.10	6.23
2	-9.65	0.09	-8.63	0.47	20	-7.21	5.21	-7.04	6.89
3	-9.31	0.15	-8.61	0.49	21	-7.14	5.84	-6.99	7.54
4	-9.03	0.24	-8.57	0.52	22	-7.12	6.03	-6.89	8.86
5	-8.45	0.64	-8.39	0.71	23	-7.04	6.86	-6.85	9.50
6	-8.36	0.75	-8.33	0.78	24	-6.97	7.80	-6.82	10.02
7	-8.24	0.92	-8.04	1.29	25	-6.87	9.18	-6.70	12.25
8	-8.17	1.03	-7.98	1.42	26	-6.83	9.88	-6.68	12.63
9	-8.08	1.19	-7.97	1.43	27	-6.67	12.85	-6.55	15.93
10	-7.97	1.43	-7.85	1.77	28	-6.58	15.09	-6.51	16.89
11	-7.82	1.85	-7.46	3.42	29	-6.56	15.61	-6.43	19.51
12	-7.75	2.08	-7.45	3.43	30	-6.51	17.05	-6.35	22.31
13	-7.62	2.58	-7.35	4.08	31	-6.37	21.47	-6.16	30.70
14	-7.55	2.91	-7.34	4.19	32	-6.14	31.71	-6.10	33.87
15	-7.49	3.22	-7.31	4.41	33			-5.81	54.83
16	-7.43	3.56	-7.30	4.47	34			-5.72	63.39
17	-7.33	4.22	-7.20	5.26	35			-4.98	225.43
18	-7.28	4.64	-7.10	6.22

• Ki^e- estimated minimum inhibitor concentration

On the other hand, the lowest binding energy and inhibitor concentration for boceprevir were −9.41 kcal.mol^-1 and 0.13 µM for the mutant M^pro, respectively. For the conformation at this binding energy, boceprevir formed a hydrogen bond with the mutant M^pro between the CYS145:N and Boc:O1 residues, between the Cys145:SG and Boc:N11 residues, and between the Thr26:O and Boc:N37 residues. Boceprevir formed a C-H bond with the mutant M^pro between the His164:O and Boc:C19 residues. Boceprevir formed alkyl interactions between the Met49 and Met165 residues and pi-alkyl interactions between the His41 and His163 residues (Figure 3).

Unlike the wild-type M^pro-boceprevir interaction, it was observed that boceprevir did not interact with the mutant type, as shown by the absence of hydrogen bonding with His41 and alkyl interaction with Pro168. A well-regulated water molecule appears to stabilize the existing covalent conformation in the binding pocket in both wild and mutant protein-boceprevir interaction by coordinating hydrogen bonds between the amide group of α-ketoamide and the backbone oxygen of Thr26.

The Debye-Waller factors calculated by elastic network model analysis were 0.58 and 0.64 Ų for the wild-type M^pro and mutant M^pro, respectively. Although the collective motion (Figure 4) of the protein did not change, the fluctuation distances decreased. The root mean square fluctuation (RMSF) (Figure 5) and flexibility values (Table 2) of residues showed that the protein transformed into a rigid structure after the mutations.

Figure 2
Bond interaction of boceprevir with wild-type SARS-CoV-2 M^pro. a) Surface representation, b) Diagram representation b)

Figure 3
Bond interaction of boceprevir with mutant SARS-CoV-2 M^pro. a) Surface representation, b) Diagram representation

DISCUSSION

M^pro cleaves polypeptide sequences after a glutamine residue, positioning the main protease as an ideal drug target because no human host-cell proteases are known with this substrate specificity [⁵⁹]. The SARS-CoV-2 M^pro consists of three basic domains. Domain I (residues 8-101) and domain II (residues 102-184) form a β-barrel structure. Domain III (residues 201-306) consists of α-helices. Domains II and III are connected by a long loop (residues 185-200). Located in a cleft between domains I and II, the active site of M^pro contains a catalytic pair of conserved Cys145 and His41 residues [²²]. M^pro domains I and III are effective in functional movements and can undergo major structural changes.

Figure 4
Slow mode collective motion of SARS-CoV-2 M^pro by Anisotropic network model. Vector ends indicate the direction of motion. Red represents mobile residues, white represents less mobile residues, white represents non-mobile residues. a) Pose-1 of wild-type M^pro, b) Pose-2 of wild-type M^pro, c) Pose-1 of mutant M^pro, d) Pose-2 of mutant M^pro

Figure 5
Root mean square fluctuations of residues by Gausian network model. a) Wild-type SARS-CoV-2 M^pro, b) Mutant SARS-CoV-2 M^pro

Boceprevir is an important protease inhibitor candidate for the SARS-CoV-2 M^pro. In this study, we revealed that seven mutations detected in the M^pro of SARS-CoV-2 caused a decrease in the binding affinity of boceprevir. The mutations caused changes in the dynamic structure of M^pro and binding affinity of the inhibitor. The change in the bond number and formation with boceprevir in the mutant M^pro substrate binding pocket and catalytic region resulted in a decrease in affinity. It was observed that hydrogen bonding between boceprevir and the His41 residue in the catalytic region of the enzyme did not occur in the mutant M^pro (Figures 2 and 3). The absence of Pro168-boceprevir hydrophobic alkyl interaction in the substrate binding pocket in the mutant protein affected the binding pattern. Khan et al. (2020) revealed that the Arg60Cys mutation detected in the M^pro in the Vietnam isolates of SARS-CoV-2 may cause a change in the dynamic structure of M^pro and a decrease in inhibitor affinity. Substitution of an amino acid with a charged side chain to an uncharged cysteine residue leads to loss of conserved ionic bond interaction, and the effect cascades to other conserved ionic interactions [¹⁰]. The interaction of boceprevir with the two catalytic residues (His41 and Cys145) of the enzyme is important for the inhibition of catalytic activity. Boceprevir interaction with the His163 and His164 residues in the substrate binding pocket, which plays an important role in catalytic activity, appears to exist in both wild-type and mutant M^pro. The RMSF results showed the effect of mutations on dynamic fluctuations of residues. It was observed that fluctuation intervals were decreased in the mutant protein, while collective molecular fluctuation was preserved. Substrate recognition regions tend to exhibit structural flexibility to mediate substrate specificity [⁶⁰]. It is thought that immobilization of key substrate binding site residues (His163, His164, Met165, and Glu166) and flexibility of residues (Ala191) near the binding site contribute to the realization of the bending (hinge) action for enzymatic activity [²²,²⁴,⁶¹,⁶²]. Post-mutation reduction in flexibility at Pro108Ser (hinge region) and Ala191Val (near hinge, flexible region) residues may affect substrate binding and enzymatic activity (Table 2). The contribution of mutations in these two residues to the decreased ligand affinity is possible.

The Glu166 residue plays a role in dimerization, which is important for the catalytic activity of the enzyme. The NH2-terminal of each of the two protomers of the M^pro interacts with the Glu166 of the other protomer and helps shape the S1 pocket of the substrate binding site and oxyanion hole [⁶¹,⁶³-⁶⁵]. In this study, it was observed that mutations transform the secondary structure between the 165 and 167 residues from coil to beta sheet. This change in the secondary structure may also affect dynamic fluctuation for Glu166 (B-factor: 24.457-0.883 Ų). It is thought that the changes caused by mutations may affect the enzyme’s active-inactive conformational transformation and rearrangements in the substrate binding pocket.

The distance between His41:NE2 and Cys145:SG in the catalytic region in the mutant M^pro was positionally opened at 0.1 Å. Similarly, the distance between His41:CE1 and His164:O opened at 0.2 Å. In the mutant M^pro, the distance between His41:ND1 and His164:ND1 narrowed from 4.3 to 4.2 Å. The distance between His41:NE2 and Boc:O04 expanded from 2.7 Å in wild-type M^pro to 3.6 Å in mutant M^pro (Figure 6). After these positional changes, it was observed that the H bond interaction between His41 and boceprevir disappeared in the mutant M^pro.

Figure 6
Hydrogen bond interaction of boceprevir with catalytic His41 residue of SARS-CoV-2 M^pro. a) Wild-type SARS-CoV-2 M^pro , b) Mutant SARS-CoV-2 M^pro

Cys145 is known to play a role in the autocleavage of the M^pro. The sulfur atom of the nucleophile Cys145, covalently bonded to the carbonyl carbon atom of the Gln306 residue in the C-terminal region, carries out autocleavage activity [⁶⁵-⁶⁸]. The preservation of the hydrogen bond between Cy145-Boc in both wild-type and mutant proteins despite the reduction in boceprevir affinity provides data that autocatalytic activity is inhibited and the conversion of M^pro to the dimer can be prevented.

Gly143 and His41 are the residues that are located in the M^pro binding pocket which is responsible for substrate stability by the hydrogen bonds they form [²⁸,³¹]. While Gly143 hydrogen bond interaction is present in the wild protein, this interaction seems to disappear after mutations. It is seen that the hydrogen bond seen between His41 and boceprevir in the wild protein transforms into the pi-alkyl interaction, which is a weaker form of interaction. The change in the bond interaction in these 2 residues that provide protease-boceprevir complex stabilization may be responsible for the decrease in boceprevir affinity.

The electrophilic α-ketoamide carbonyl appears to form a covalent bond with the sulfur of the catalytic Cys145 and an S,O-acetale is formed. In Wild-type M^pro, α-ketoamide oxygen forms hydrogen bonds with the main chain amides of Cys145 and Gly143 that occupy the oxyanion hole [²⁸,⁶⁹]. Herein, it is His41 that controls all congruence. In the mutant protein, it is seen that the hydrogen bonding interaction of Gly143 is eliminated and the formation is disrupted. The elimination of the Gly143 interaction occupying the oxyanion hole and the weakening of His41's interaction may affect the catalytic protonation of His41 and Cys145 residues. It is possible that the catalytic activity of M^pro in its natural form, which is blocked by boceprevir, may be restored in the mutant protein and show catalytic activity, albeit reduced.

CONCLUSION

An increasing number of cases due to transmission from person to person will cause new mutations to occur. The mutations in the SARS-CoV-2 M^pro can have negative consequences, such as a high increase in catalytic activity or an undesirable decrease in inhibitor affinity. Each new mutation carries risks that will invalidate therapies developed specifically for important viral biological structures. In this study, it was determined that SARS-CoV-2 M^pro mutations can cause a decrease in the affinity of boceprevir, one of the important therapeutic inhibitors for COVID-19 treatment. The results of this study will provide important data for drug design studies targeting the SARS-CoV-2 M^pro. However, further in vitro and in vivo analyses around this mentioned compound are required to ascertain these suggestions.

Acknowledgment

The genome and proteome sequence data of SARS CoV-2 were obtained from Virus database of National Center Biotechnology Information.

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• Funding:

  This research received no external funding.

Edited by

Editor-in-Chief:

Alexandre Rasi Aoki

Associate Editor:

Daniel Fernandes

Publication Dates

• Publication in this collection
  21 Feb 2022
• Date of issue
  2021

History

• Received
  23 Dec 2020
• Accepted
  25 May 2021