Discovery of Dithioacetal Derivatives Containing Sulfonamide Moiety of Novel Antiviral Agents by TMV Coat Protein as a Potential Target.

Tobacco mosaic virus coat protein (TMV CP) plays an important role in viral replication, translation, and intracellular and intercellular movements. Thus, TMV CP could be regarded as a potential target for antiviral agents. In this study, in order to find out whether dithioacetal derivatives act on the CP target, a series of dithioacetal derivatives containing sulfonamide moiety was first designed and synthesized. Bioassay results demonstrated that Y14, Y18, and Y21 exhibited excellent activities against TMV, with half-maximal effective concentrations (EC50) of the curative, protective, and inactivate activities being 183.0 ± 3.2, 252.3 ± 2.6, and 63.8 ± 1.2 μg/mL, 270.6 ± 3.7, 249.7 ± 3.5, and 57.7 ± 1.4 μg/mL, and 329.5 ± 1.5, 269.2 ± 3.7, and 48.1 ± 2.0 μg/mL for Y14, Y18, and Y21, respectively, which were higher than those for the control agents ningnanmycin (331.0 ± 2.8, 271.0 ± 2.8, and 77.4 ± 1.3 μg/mL, respectively) and d2 (471.5 ± 1.4, 447.2 ± 2.1, and 91.7 ± 1.8 μg/mL, respectively). Transmission electron microscopy showed that the particle morphology of TMV was destroyed by Y21, and microscale thermophoresis (MST) showed that Y21 bonded to CP with a dissociation constant (K d) of 9.7 ± 1.7 μM. Then, molecular docking and MST further illustrated that Y21 had a weak binding affinity with the TMV mutant protein (K d = 561.3 ± 83.2 μM). Thus, we deduced that the dithioacetal derivative Y21 may inhibit TMV activity by binding TMV CP. This work provides some new insights for the design and optimization of novel anti-TMV agents.

Introduction

Tobacco mosaic virus (TMV) is the cause of devastating diseases in major crops, including vegetables and tobacco. These diseases usually bring about severe damage and enormous financial losses;[1,2] however, there are no prominent management and treatment methods available to shelter plants from TMV infection nowadays.[3] The scarcity of valid antiviral agents is largely due to insufficient knowledge of the underlying targets connected with the TMV infection. TMV coat protein (CP) plays an important role in the self-assembly of TMV through an initial RNA recognition reaction, and it is of great necessity for virus assembly initiation and elongation in tobacco plants.[4] So, we can regard TMV CP as a potential target for the commercial antiviral agents;[5] however, few studies have focused on viral CP as potential targets for antiviral agent discovery.

Ningnanmycin (Figure ), a most effective commercial antiviral agent, belongs to microbial pesticides and targets TMV CP.[6] Compared with other existing commercial formulations, it is more effective in the treatment for TMV. However, the use of this antiviral agent for field trial is mainly limited by its photosensitivity and water stickiness.[7] Ribavirin (Figure ) is another widely used inhibitor of plant viruses, but its inhibitory effect is undesirable.[8] Therefore, developing an efficient, low-toxic, and environmentally friendly antiviral agent through chemical synthesis has become an urgent area for eliminating the attack of TMV. Based on the above mentioned functions of TMV CP, we can consider TMV CP as a potential target for viral inhibitors.

Figure 1

Chemical structures of ribavirin, ningnanmycin, and d2.

Sulfonamides act as plant immune-priming compounds that potentiate pathogen-induced cell death and increased disease resistance in Arabidopsis plants. Sulfonamides and dithioacetal derivatives have a broad spectrum of biological activities, such as weeding,[9] insecticidal,[10,11] antifungal,[12] anti-HIV,[13,14] anticancer,[15] antiplant virus,[16−19] herbicide,[20] bactericidal,[21] antitumor,[22] and antiviral.[23−26] Hence, their synthesis and derivatives have attracted extensive attention from the organic and pharmacological chemical community.

Our research group reported for the first time that dithioacetal derivatives exert good curative and protective activities against TMV.[23,24] Then, we found that the sulfonamide derivatives d2 had good inactivation activity against TMV through activity screening.[17] However, the inactivation activities of these compounds against TMV and their targets to TMV remain unclear. In this work, sulfonamide moiety was introduced into the dithioacetal through the method of active methylene stitching, and a series of novel dithioacetal derivatives containing sulfonamide moiety was first designed and synthesized (Figure ), and the activities of the target compounds against TMV in vivo and in vitro were methodically evaluated, followed by the binding affinity of the title compound with TMV CP, molecular docking, and site-directed mutation. This work provides some new insights into the design, synthesis, and optimization of novel anti-TMV agents based on TMV CP as a molecular target.

Figure 2

Design of target compounds.

Results and Discussion

Chemistry

Scheme presents the synthetic routes of the dithioacetal derivatives containing the sulfonamide moiety. First, bromine ethylamine hydrobromide was added to different substituted benzenesulfonyl chlorides in dichloromethane (DCM) using Et3N as a catalyst to obtain intermediates a1–a9. Then, solutions of a1–a9 in MeCN were added to substituted hydroxybenzaldehyde, with KI and K2CO3 as catalysts to get the intermediates b1–b13. Finally, b1–b13 were added to substituted thiols in DCM using SiO2·NaHSO4 as a catalyst to receive the title compounds Y1-28 (Scheme ). The structures of the intermediates and title compounds were characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, and 19F NMR spectroscopies, high-resolution mass spectrometry (HRMS), and Fourier transform infrared spectroscopy (FT-IR) (see the Supporting Information for details).

Scheme 1

Design and Synthetic Routes of Intermediates and Title Compounds

Antiviral Activities In Vivo

The antiviral activities of the intermediates and title compounds against TMV are shown in Tables –3. Bioassay results demonstrated that the intermediates a1–a9 and b1–b13 exhibited good antiviral activities but lower than those of the target compounds, and Y14, Y18, and Y21 exhibited excellent activities against TMV. The half-maximal effective concentration (EC50) of the curative, protective, and inactivate activities were 183.0 ± 3.2, 252.3 ± 2.6, and 63.8 ± 1.2 μg/mL for Y14, 270.6 ± 3.7, 249.7 ± 3.5, and 57.7 ± 1.4 μg/mL for Y18, and 329.5 ± 1.5, 269.2 ± 3.7, and 48.1 ± 2.0 μg/mL for Y21, respectively, which were higher than those for the control agents ningnanmycin (331.0 ± 2.8, 271.0 ± 2.8, and 77.4 ± 1.3 μg/mL, respectively) and d2 (471.5 ± 1.4, 447.2 ± 2.1, and 91.7 ± 1.8 μg/mL, respectively).

Table 1

Antiviral Activities of Target Compounds Y1–Y28 against TMV at 500 μg/mLa,d

compound	curative activitya (%)	EC50 for curative activitya (μg/mL)	protective activitya (%)	EC50 for protective activity (μg/mL)	inactivate activitya (%)	EC50 for inactivate activitya (μg/mL)
Y1	48.0 ± 4.9	998.5 ± 2.1	53.1 ± 4.1	155.1 ± 1.3	48.3 ± 4.9	515.0 ± 0.6
Y2	52.8 ± 0.6	258.5 ± 2.3	64.6 ± 4.4	597.1 ± 1.7	52.0 ± 4.6	612.6 ± 0.7
Y3	53.9 ± 5.0	1481.4 ± 2.5	53.2 ± 3.4	174.3 ± 2.1	43.0 ± 3.3	521.9 ± 0.9
Y4	46.4 ± 4.1	110.9 ± 4.3	42.8 ± 2.2	176.8 ± 2.3	47.7 ± 3.6	566.5 ± 2.2
Y5	19.9 ± 4.6	c	39.8 ± 4.3	654.5 ± 3.2	61.4 ± 1.6	156.5 ± 0.8
Y6	50.0 ± 4.5	356.8 ± 3.5	45.2 ± 4.1	567.9 ± 4.6	54.6 ± 0.4	210.5 ± 1.4
Y7	17.5 ± 1.3	c	31.5 ± 4.1	892.2 ± 2.3	55.8 ± 0.4	411.9 ± 0.7
Y8	10.9 ± 2.3	c	62.2 ± 4.0	320.0 ± 3.2	25.8 ± 2.2	1232.3 ± 0.4
Y9	38.6 ± 3.7	764.9 ± 4.1	42.0 ± 3.2	636.1 ± 3.2	52.2 ± 3.8	495.3 ± 1.8
Y10	54.1 ± 1.5	101.6 ± 2.8	36.0 ± 4.4	672.3 ± 3.2	72.7 ± 3.7	133.8 ± 1.8
Y11	56.4 ± 4.8	432.4 ± 3.8	76.5 ± 2.3	329.9 ± 3.8	65.8 ± 4.6	262.9 ± 1.6
Y12	53.4 ± 3.7	243.9 ± 2.5	51.0 ± 4.3	307.7 ± 2.7	15.5 ± 3.0	2003.5 ± 1.7
Y13	24.8 ± 2.0	c	41.6 ± 4.1	339.5 ± 4.3	71.5 ± 2.1	43.2 ± 2.3
Y14	56.3 ± 1.5	183.0 ± 3.2	53.3 ± 1.6	252.3 ± 2.6	78.7 ± 2.1	63.8 ± 1.2
Y15	61.2 ± 4.9	322.1 ± 2.6	61.5 ± 3.8	305.8 ± 2.0	42.2 ± 2.4	1546.8 ± 0.8
Y16	13.5 ± 3.9	c	61.9 ± 4.3	200.7 ± 1.9	23.0 ± 4.2	771.6 ± 1.2
Y17	53.2 ± 2.9	463.6 ± 4.8	53.2 ± 2.9	210.6 ± 4.2	66.7 ± 4.7	164.5 ± 1.4
Y18	56.8 ± 4.5	270.6 ± 3.7	59.8 ± 4.4	249.7 ± 3.5	73.9 ± 1.2	57.7 ± 1.4
Y19	60.3 ± 0.1	479.3 ± 1.3	60.3 ± 0.1	312.4 ± 3.1	59.2 ± 1.3	42.4 ± 0.76
Y20	46.8 ± 1.9	295.8 ± 3.5	50.8 ± 1.5	515.2 ± 4.3	87.7 ± 4.7	79.0 ± 1.3
Y21	59.5 ± 0.4	329.5 ± 1.5	69.3 ± 3.7	269.2 ± 3.7	87.2 ± 3.6	48.1 ± 2.0
Y22	50.7 ± 1.3	215.0 ± 2.9	48.0 ± 1.8	407.5 ± 2.6	24.7 ± 1.7	c
Y23	44.7 ± 3.6	572.1 ± 3.2	55.6 ± 2.2	563.7 ± 1.6	53.2 ± 4.3	383.2 ± 3.1
Y24	19.6 ± 1.1	c	40.2 ± 3.3	631.1 ± 2.9	18.7 ± 2.4	1066.3 ± 4.3
Y25	60.8 ± 3.0	383.3 ± 0.9	58.3 ± 3.1	271.3 ± 1.3	24.5 ± 1.7	766.7 ± 4.3
Y26	27.2 ± 2.7	c	54.6 ± 5.0	1063.4 ± 1.8	29.7 ± 2.4	856.2 ± 4.5
Y27	53.2 ± 1.2	393.5 ± 7.5	53.5 ± 1.3	287.7 ± 1.6	53.5 ± 2.6	495.0 ± 2.3
Y28	25.9 ± 4.6	c	58.0 ± 2.9	578.7 ± 1.7	20.1 ± 0.7	936.7 ± 2.7
ningnanmycinb	54.9 ± 4.9	331.0 ± 2.8	65.4 ± 2.1	271.0 ± 2.8	85.4 ± 2.8	77.4 ± 1.3
d2b	51.1 ± 1.7	471.5 ± 1.4	52.2 ± 1.3	447.2 ± 2.1	71.8 ± 2.3	91.7 ± 1.8

Average of three replicates.

Ningnanmycin and d2 were used as positive controls.

Increasing the concentration of the title compound to 2500 μg/mL still does not reach 50% of the prevention effect against TMV.

The ± values represent standard deviation.

Table 3

Antiviral Activities of Intermidates a1–a9 and b1–b13 Against TMV at 500 μg/mLc

compound	inactivate activitya (%)	compound	inactivate activitya (%)
a1	60.0 ± 4.7	b1	13.9 ± 2.6
a2	47.8 ± 4.7	b2	27.2 ± 4.7
a3	25.5 ± 3.5	b3	46.5 ± 4.6
a4	55.9 ± 4.4	b4	45.7 ± 4.6
a5	51.2 ± 4.6	b5	40.3 ± 4.9
a6	55.2 ± 3.3	b6	32.9 ± 3.7
a7	52.8 ± 4.9	b7	43.6 ± 1.3
a8	50.7 ± 2.6	b8	55.0 ± 3.0
a9	62.2 ± 4.7	b9	59.8 ± 2.7
		b10	39.7 ± 2.0
Y21	87.2 ± 3.6	b11	42.6 ± 4.9
d2b	71.8 ± 2.3	b12	41.1 ± 4.8
ningnanmycinb	83.2 ± 4.7	b13	66.1 ± 4.9

Average of three replicates.

Ningnanmycin and d2 were used as positive controls.

The ± values represent standard deviation.

The sulfonamide structural unit introduced into the dithioacetal structure and a series of dithioacetal derivatives containing the sulfonamide moiety were first designed and synthesized. Bioassay results revealed that the target compounds Y5, Y6, and Y7 have good inactivate activities, but compared with Y1–Y4, the activity changes were not very significant, indicating that changes in R2 and R3 will cause a slight change in activity. The general trend is that when R1 and R2 are the same, the inactivate activities of those compounds decreased gradually with propyl, isopropyl, and ethyl. Then, we studied whether R1 has an effect on the activity of Y9–Y20, and the bioassay results showed that compound Y20 had better curative, protective, and inactivate activities. In order to further synthesize highly active compounds, Y21 was synthesized. Compared with other compounds, Y21 was found to have excellent curative, protective, and inactivate activities. Finally, the target compounds Y22–Y28 were synthesized by introducing fluorine atoms, and it was found that their activities were lower than that of Y21.

The structure–activity relationships of the compounds Y1–Y28 against TMV are as follows: the type and position of the substituents of the title compounds have a great influence on the inhibitory activities against TMV. The following observations can be made from Table : (a) When the substituents R1 and R2 are the same while R3 is different, the inhibitory activities against TMV are changed a little, such as Y1 (R1 = 4-CH3, R2 = H, R3 = Et) and Y3 (R1 = 4-CH3, R2 = H, R3 = i-Pr), Y19 (R1 = 4-F, R2 = 3-OCH3, R3 = i-Pr) and Y21 (R1 = 4-F, R2 = 3-OCH3, R3 = n-Bu); (b) when R1 and R3 are the same while R2 is different, the activities of the title compounds against TMV decrease gradually with R3 = 3-OCH3, 3-Cl, such as Y15 (R1 = 3-Br, R2 = 3-OCH3, R3 = n-Pr) >Y12(R1 = 3-Br, R2 = 3-Cl, R3 = n-Pr) and Y14 (R1 = 4-Br, R2 = 3-OCH3, R3 = i-Pr) > Y10 (R1 = 4-Br, R2 = 3-Cl, R3 = i-Pr); (c) when R2 and R3 are the same while R1 is different, the compound’s effects against TMV change greatly, such as Y21 (R1 = 4-F, R2 = 3-OCH3, R3 = n-Bu) ≫ Y22 (R1 = 2,6-di-F, R2 = 3-OCH3, R3 = n-Bu) and Y14 (R1 = 4-Br, R2 = 3-OCH3, R3 = i-Pr) ≫ Y16(R1 = 3-Br, R2 = 3-OCH3, R3 = i-Pr) > Y24 (3-Cl,4-F, R2 = 3-OCH3, R3 = i-Pr). In short, the difference in R1 is the main factor of the title compounds that affects the inhibitory activities against TMV, and the changes in R3 and R2 assist R1 to change their activities.

Effect of Y21 on TMV Morphology

The effect of Y21 on the TMV morphology was observed via transmission electron microscopy (TEM). The results showed that the effects of Y21 (Figure B), ningnanmycin (Figure C), and d2 (Figure D) on the TMV morphology were greater compared to the Control Check, after mixing 1% Tween aqueous solution with an equal volume of TMV virus solution and incubating for 30 minutes, the mixture was adsorbed on a 200-mesh copper mesh carbon carrier film and counterstained with 1% phosphotungstic acid with a pH of 7.4 for 30 s. After being completely dried, the FEI Talos F200C instrument was used to observe the morphology of the TMV particles under a TEM at 200 kV (Figure A). The virions of CK were intact, long, and rod-shaped, while the virions of Y21, ningnanmycin, and d2 were severely broken, and the extent of fragmentation of Y21 was remarkably greater compared with those of ningnanmycin and d2. In addition, the densities of the virions of Y21, ningnanmycin, and d2 were sparse compared with that of CK. These findings indicated that Y21 may disrupt the assembly of virions and lead to virions losing the ability to infect plants.

Figure 3

TEM results of CK (A), target compound Y21 (B), ningnanmincin (C), and d2 (D).

Determination of the Dissociation Constant of Y21 with TMV CP

Based on the notable inactivate activity of target compound Y21, we further verified the binding affinity between Y21 and TMV CP using microscale thermophoresis (MST). The results showed that Y21 has a micromole binding affinity with TMV CP with a dissociation constant (Kd) of 9.7 ± 1.7 μM, which was equivalent to that of ningnanmycin (9.2 ± 4.1 μM) and better than those of d2 (13.7 ± 3.8 μM), Y7 (66.1 ± 9.0 μM) and Y25 (221.4 ± 65.0 μM). The MST results were consistent with the bioassay results in vivo (Figure ).

Figure 4

MST results of Y7 (A), Y25 (B), d2 (C), Y21, (D) and ningnanmycin (E) with TMV CP and Y21 (F) with TMV mutant protein.

Molecular Docking

The TEM and MST results suggested that Y21 may act on the CP of the virions. Then, we further performed molecular docking to study the binding sites of Y21 on TMV CP, and the results indicated that Y21 was inlayed into the TMV CP activity pocket of Arg134, Glu131, Lys226, Tyr139, and Val218 (Figure A,B). In these interactions, the F of Y21 interacted with the oxygen atoms of Arg134 residues by hydrogen bonding: F···O–H = 3.4 Å. The oxygen atoms of Y21 interacted with the N–H binding of Lys226 residues via two hydrogen bonds: O···H–N = 2.9, 3.1 Å. The N–H of Y21 interplayed with the oxygen atoms of Glu131 residues via hydrogen bonding: O···H–N = 4.8 Å. The oxygen atoms of Y21 were reciprocated with the N–H binding of Arg134 residues via hydrogen bonding: O···H–N = 3.2 Å. The oxygen atoms of Y21 interplayed with the N–H binding of Gly137 residues via hydrogen bonding: O···H–N = 3.4 Å. The benzene ring of compound Y21 and Tyr139 residue formed a large π bond, and the carbon atom of compound Y21 and Val218 residue interacted through a hydrophobic bond (Figure B).

Figure 5

Molecular docking results of Y21 with TMV CP.

Determination of the Dissociation Constant of Y21 with TMV Mutant Protein

We performed molecular docking to study the binding sites of Y21 on TMV CP, and the results indicated that Y21 was inlayed into the TMV CP activity pockets of Arg134, Glu131, Lys226, Tyr139, and Val218. Then, we constructed the TMV mutant protein with R134A, E131A, K226A, Y139A, and V218A and performed MST to validate whether these amino acids are the action sites of Y21 on TMV CP. The results (Figure F) showed that the binding constant of the title compound Y21 with the TMV mutant protein was 561.3 ± 83.2 μM, which indicated that Y21 had a weak binding affinity with the TMV mutant protein. These findings illustrated that the action site of Y21 on TMV CP ranges between these five amino acids.

Conclusions

In summary, 28 novel dithioacetal derivatives containing the sulfonamide moiety were designed and synthesized based on TMV CP as the molecular target, and their antiviral activities against TMV were systematically evaluated. Bioassay results demonstrated that Y14, Y18, and Y21 exhibited excellent activities against TMV. The EC50 of curative, protective, and inactivate activities were 183.0 ± 3.2, 252.3 ± 2.6, and 63.8 ± 1.2 μg/mL for Y14, 270.6 ± 3.7, 249.7 ± 3.5, and 57.7 ± 1.4 μg/mL for Y18, and 329.5 ± 1.5, 269.2 ± 3.7, and 48.1 ± 2.0 μg/mL for Y21, respectively, which were higher than those for the control agents ningnanmycin (331.0 ± 2.8, 271.0 ± 2.8, and 77.4 ± 1.3 μg/mL, respectively) and d2 (471.5 ± 1.4, 447.2 ± 2.1, and 91.7 ± 1.8 μg/mL, respectively). Based on the prominent inactivate activity of Y21, we performed TEM and MST to observe its effect on TMV; the TEM and MST results suggested that Y21 may act on the CP of the virions. Then, we conducted molecular docking to study the binding sites of Y21 on TMV CP, and the result showed that Y21 was inlayed into TMV CP activity pockets of Arg134, Glu131, Lys226, Tyr139, and Val218. Finally, site-directed mutation was proceeded to further investigate whether these five amino acid residues are the possible sites of action for compound Y21 on TMV CP. The MST results demonstrated that compound Y21 had a weak binding affinity with the TMV mutated protein with a dissociation constant of 561.3 ± 83.2 μM. This result directly certified that the five amino acid residues may be the key action sites of Y21 on TMV CP. These above mentioned findings provide some important insights for the design of highly active compounds and further research into their mechanism of action.

Materials and Methods

Chemicals and Instruments

All of the reagents and solvents were purchased from commercial suppliers and were used without further purification and drying. The melting points of the intermediates and title compounds were measured on a WRX-4 melting point apparatus with a binocular microscope (Shanghai YiCe Apparatus & Equipment Co., Ltd., China). The progress of the reactions was tracked by thin-layer chromatography (TLC) on silica gel GF254 and identified by UV. 1H and 13CNMRspectra were obtained with a Bruker DPX 400 MHz (Bruker, Germany) system in CDCl3. Chemical shifts (δ) were measured in parts per million, and tetramethylsilane was used as an internal standard. HRMS was performed with a Fourier transform ion cyclotron resonance mass spectrometer (Varian, Palo Alto, CA, USA). FT-IR was recorded on the FT-IR spectrometer (Nicolet iS50).

General Procedure for the Preparation of Intermediates a1–a9(27)

A solution of bromoethylamine hydrobromide (13.6 mmol) in CH2Cl2 (25 mL) was dropwise added to Et3N (11.5 mmol) and stirred for 30 min at room temperature. Different substituted benzenesulfonyl chlorides (10.5 mmol) were dissolved in CH2Cl2 and added to the reaction system slowly, and each mixture was observed at room temperature for 8–10 h via TLC. Then, the mixtures were added to diluted hydrochloric acid to adjust the pH to 1–2 and extracted three times with 50 mL of CH2Cl2. The organic layer was dried over Na2SO4 (s), and the solvent was removed under reduced pressure. The obtained residues were recrystallized using DCMand petroleum ether to acquire intermediates a1–a9 (Scheme ).

General Procedure for the Preparation of Intermediates b1–b13(28)

Solutions of a1–a9 (1.5 mmol) were added to substituted 4-hydroxybenzaldehyde (1.1 mmol) in MeCN (25 mL), with KI (112.1 μmol) and K2CO3 (1.2 mmol) as catalysts, stirring for 4–6 h at 80 °C to get the reaction completed (indicated by TLC). Then, the mixtures were filtrated and concentrated to get the crude intermediates b1–b13, which were further purified by column chromatography on SiO2 gel with petroleum ether/ethyl acetate (5:1, v/v) as the eluent to obtain the intermediates b1–b13 (Scheme ).

General Procedure for the Preparation of Title Compounds Y1–Y28(23−26)

Solutions of b1–b13 (597.1 μmol) and SiO2·NaHSO4 (597.1 μmol) in CH2Cl2 (20 mL) were slowly added to different substituted thiols (2.4 mmol), stirred, and refluxed for 4–6 h until completion of the reaction. Then, they were filtrated and concentrated to receive the crude target compounds Y1–Y28, which were further purified by column chromatography on SiO2 gel using petroleum ether/ethyl acetate (5:1, v/v) as the eluent to gain the title compounds Y1–Y28 (Scheme ).

Antiviral Activity Assay

TMV extraction was performed according to a previously reported method.[29]

Evaluation of Anti-TMV Activities In Vivo

The curative, protective, and inactivate modes of the target compounds against TMV in vivo were performed according to the method reported in the literature.[30] All tests were replicated three times.

Evaluation of Anti-TMV Activities In Vitro

The impacts of the title compounds on the TMV morphology were evaluated by TEM.[31,32] The binding forces of the title compounds with TMV CP and a quintuple mutant protein were analyzed by using MST to obtain the dissociation constant (Kd).[33,34] Ningnanmycin and d2 were used as positive controls, and all tests were repeated three times.

Molecular Docking

Molecular docking is a computer-assisted method that can be used to study the interactions between the receptor and the ligand and has been successfully used in drug design.[35] Autodock is a well-known docking software,[36,37] and the interaction mode of Y21 with TMV CP was studied by using Autodock 4.0 software.[38]

Table 2

Regression Equation of Y14, Y18, Y21, d2, and Ningnanmycinc

compound	Y14	Y18	Y21	d2b	ningnanmycinb
EC50 of curative activitya	183.0 ± 3.2	270.6 ± 3.7	329.5 ± 1.5	471.5 ± 1.4	331.0 ± 2.8
regression equation	y = 0.56x + 3.73	y = 1.12x + 2.26	y = 1.43x + 1.40	y = 0.61x + 3.37	y = 0.75x + 3.11
R	0.97	0.94	0.97	0.93	0.98
EC50 of protective activitya	252.3 ± 2.6	249.7 ± 3.5	269.2 ± 3.7	447.2 ± 2.1	271.0 ± 2.8
regression equation	y = 1.22x + 2.08	y = 1.18x + 2.16	y = 1.02x + 2.52	y = 0.71x + 3.12	y = 0.82x + 3.00
R	0.92	0.97	0.98	0.99	0.99
EC50 of inactivate activitya	63.8 ± 1.2	57.7 ± 1.4	48.1 ± 2.0	91.7 ± 1.8	77.4 ± 1.3
regression equation	y = 0.52x + 4.06	y = 0.41x + 4.28	y = 0.97x + 3.37	y = 0.68x + 3.66	y = 1.55x + 2.08
R	0.99	0.99	0.96	0.98	0.96

Average of three replicates.

Ningnanmycin and d2 were used as positive controls.

The ± values represent standard deviation.