Tobacco mosaic virus (TMV); coat protein (CP)

Identifying that Ningnanmycin (NNM) binds to TMV CP and TMV RNA[1]
Recombinant CP four-layer aggregate discs were formed in phosphate buffer at 20°C for more than 12 h. They were collected as target proteins and TMV RNA was isolated from infected-TMV tobacco leaves. The TMV RNA was added to the recombinant CP four-layer aggregate discs to form TMV particles. The recombinant CP exhibited an activity similar to its native form in the construction of infectious viral particles [6]. We studied the interactions between NNM and TMV particles, NNM and CP discs, and TMV RNA and CP discs using microscale thermophoresis (MST) (Figure ​(Figure1A).1A). The NNM bound to TMV particles with a dissociation constant (Kd) of 25.8–52.3 μM (Figure ​(Figure1B1B and Supplementary Table 1), the NNM bound to CP discs with a Kd of 1.10–3.96 μM (Figure ​(Figure1C1C and Supplemental Table 1), and the TMV RNA bound to CP discs with a Kd of 144.8–207.3 μM from 20°C to 30°C (Figure ​(Figure1D1D and Supplemental Table 1). The affinity between NNM and CP discs or TMV particles was greater than the affinity between TMV RNA and CP discs. Additionally, we studied the interactions between NNM and TMV RNA using isothermal titration calorimetry (ITC) (Figure ​(Figure1E).1E). The NNM bound to TMV RNA with a Kd of 16.5 μM (Figure ​(Figure1E1E and Supplemental Table 1). The affinity between NNM and CP discs was greater than the affinity between NNM and TMV RNA. Thus, the target of NNM is main CP discs. Because TMV particles are composed of TMV CP discs and TMV RNA, and TMV CP discs are stabilized by numerous protein–protein salt bridges and hydrogen-bonding networks among protein layers, we reasoned that NNM bound CP with a high affinity through similar chemical bonds by competing for the residues contributing to disc stability. Thus, it may be possible to destabilize the CP complex and decrease the virulence.

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NNM induces the disassembly of TMV CP discs[1]
NNM was capable of disrupting the oligomeric structure of recombinant CPs of TMV. Briefly, recombinant TMV CP was allowed to form functional aggregates. After adding the test compounds, the protein samples were examined by size-exclusion chromatography (SEC) and transmission electron microscopy (TEM) (Figure ​(Figure2).2). We further analyzed the effects of these compounds on the biochemical properties of this CP. As expected, initially all recombinant TMV CP was eluted as discs (Figures 2A–B). When NNM was added at a ratio of 1:5 (CP four-layer aggregate discs:NNM molecules) to the disc solution, a significant portion (60%) of the discs disassembled into monomeric proteins (Figure ​(Figure2C).2C). The induction of disc disassembly by NNM was dose-dependent. Increasing the amount of NNM led to a larger extent of disassembly. When the ratio between TMV CP and NNM reached 1:20, the discs were completely disassembled into monomeric proteins (Figures 2D–E). Additionally, the wild type (wt) CP four-layer aggregate discs (isolated from TMV particles) were used as targets, to test the interaction between the wt CP four-layer aggregate discs and NNM. When the ratio between the wt CP four-layer aggregate discs and NNM molecules reached 1:20, the wt CP four-layer aggregate discs were completely disassembled into monomeric proteins (Supplementary Figure 1). The affinities between NNM–CP monomers and CP–CP monomers were evaluated, and the results implied that NNM have a strong affinity of 18.6 μM, leading to CP disc disassembly (the Kd of CP–CP was 234.2 μM) (Figures 3A–3C). We thus deduced that NNM disrupts the uniform multimeric structure of the CP.

Evaluating Ningnanmycin (NNM) ability to inhibit TMV replication in vivo[1]
To assess whether NNM can inhibit TMV CP replication in a systemic infection of the host Nicotiana tabacum cv. K326 (N. tabacum cv. K326), the leaf disc method along with western blot analysis, of TMV CP exposed to 0.5 mg/mL NNM for 24–96 h were carried out. The bands of the CP were weak when sprayed with 0.5 mg/mL NNM for 24 h and the bands of the CP were not visible after 72 h (Figure ​(Figure8A),8A), which indicated that the newly assembled TMV virions in tobacco were inhibited by NNM Additionally, we collected the mutated protein discs and reconstituted a protein solution of 1.7 mg/mL, which was incubated with 0.5 mg/mL TMV RNA for 30 min to reconstitute viral particles (Figure ​(Figure8B).8B). In TMV inoculation assays, the infectivity of the viruses derived from mutated CP significantly decreased (Figure ​(Figure8C).8C). Additionally, the mean lesions caused by the mutant viruses were about one quarter the size of those caused by the full-length reconstituted virus (Figure ​(Figure8D).8D). Thus, residues Ser15, Ser49, Arg71 and Tyr72, which were identified in computational simulations, indeed play key roles in the NNM–CP interactions.[1]

Verifying the interaction between Ningnanmycin (NNM) and mutated TMV CP [1]
To verify the results of the MD simulation, four mutations, S15G, S49G, R71G, and Y72G, were introduced into the CP, and the mutant proteins were used to measure the interactions between NNM and TMV CP as described previously (see interactions between NNM and TMV CP). Then, 1mL of purified self-assembled mutant discs (8.7 mg/mL) were incubated in 10 mM sodium phosphate and 100 mM sodium chloride solution, pH 7.2, and mixed with 0.2 mL purified TMV RNA (2 mg/mL). The mixture was incubated at 20°C for 24 h. Suspensions were centrifuged at 2,700 ×g for 1 min, and reconstituted viruses were obtained.

[1]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669903/

Gougerotin is a peptidyl nucleoside antibiotic which functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase. It has a role as an EC 2.3.2.* (aminoacyltransferase) inhibitor, a bacterial metabolite, a protein synthesis inhibitor, a nucleoside antibiotic, an antimicrobial agent and a fungicide. It is a triol, a pyrimidine nucleoside and an oligopeptide. It is functionally related to a cytosine and a serine.
Asteromycin has been reported in Streptomyces citricolor, Streptomyces graminearus, and other organisms with data available.
See also: Gougerotin (annotation moved to).

C16H25N7O8

443.412

443.176

156410-09-2

159643

Typically exists as solid at room temperature

1.8±0.1 g/cm3

1.745

Streptomyces noursei var

-3.55

8

9

8

31

788

0

CNCC(=O)NC(CO)C(=O)NC1C(O)C(O)C(OC1C(N)=O)n1ccc(N)nc1=O

AMNAZJFEONUVTD-UHFFFAOYSA-N

InChI=1S/C16H25N7O8/c1-19-4-8(25)20-6(5-24)14(29)22-9-10(26)11(27)15(31-12(9)13(18)28)23-3-2-7(17)21-16(23)30/h2-3,6,9-12,15,19,24,26-27H,4-5H2,1H3,(H2,18,28)(H,20,25)(H,22,29)(H2,17,21,30)

6-(4-amino-2-oxopyrimidin-1-yl)-4,5-dihydroxy-3-[[3-hydroxy-2-[[2-(methylamino)acetyl]amino]propanoyl]amino]oxane-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)