WO2021209689A1 - Compounds and compositions for treating sweet potato against sweet potato pathogenic viruses - Google Patents
Compounds and compositions for treating sweet potato against sweet potato pathogenic viruses Download PDFInfo
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- WO2021209689A1 WO2021209689A1 PCT/FI2021/050285 FI2021050285W WO2021209689A1 WO 2021209689 A1 WO2021209689 A1 WO 2021209689A1 FI 2021050285 W FI2021050285 W FI 2021050285W WO 2021209689 A1 WO2021209689 A1 WO 2021209689A1
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- 0 *C1N(*)c2c(*)c(*)c(*)c(I)c2C2C=CCC12 Chemical compound *C1N(*)c2c(*)c(*)c(*)c(I)c2C2C=CCC12 0.000 description 7
- VSRZDWAATYOPFA-UHFFFAOYSA-N Cc1ccc(C(C(CC=C2)C2c2c3)Nc2ccc3C(O)=O)cc1 Chemical compound Cc1ccc(C(C(CC=C2)C2c2c3)Nc2ccc3C(O)=O)cc1 VSRZDWAATYOPFA-UHFFFAOYSA-N 0.000 description 1
- PMIRIFAQCGEAPM-UHFFFAOYSA-N O=C(CSc1nnc[nH]1)c(cc1)cc(Cl)c1Cl Chemical compound O=C(CSc1nnc[nH]1)c(cc1)cc(Cl)c1Cl PMIRIFAQCGEAPM-UHFFFAOYSA-N 0.000 description 1
- AFUQGUYCNGJYNT-UHFFFAOYSA-N OC(c(cc1)cc(C2C=CCC22)c1NC2C1CC=CCC1)=O Chemical compound OC(c(cc1)cc(C2C=CCC22)c1NC2C1CC=CCC1)=O AFUQGUYCNGJYNT-UHFFFAOYSA-N 0.000 description 1
- LKCVPAJAXLUURH-UHFFFAOYSA-N OC(c(cc1C2C=CCC22)ccc1NC2c1ccccc1)=O Chemical compound OC(c(cc1C2C=CCC22)ccc1NC2c1ccccc1)=O LKCVPAJAXLUURH-UHFFFAOYSA-N 0.000 description 1
- AOWWBFQCNXXGFN-UHFFFAOYSA-N OC(c1ccc(C(C(CC=C2)C2c2ccc3)Nc2c3Br)cc1)=O Chemical compound OC(c1ccc(C(C(CC=C2)C2c2ccc3)Nc2c3Br)cc1)=O AOWWBFQCNXXGFN-UHFFFAOYSA-N 0.000 description 1
- FADYQKSIESMOII-UHFFFAOYSA-N Oc1ccc(C(N2)Nc3cccc4c3c2ccc4)cc1 Chemical compound Oc1ccc(C(N2)Nc3cccc4c3c2ccc4)cc1 FADYQKSIESMOII-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/16—Ring systems of three rings containing carbocyclic rings other than six-membered
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N41/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom
- A01N41/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom containing a sulfur-to-oxygen double bond
- A01N41/04—Sulfonic acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
- A01N43/42—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings condensed with carbocyclic rings
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/64—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with three nitrogen atoms as the only ring hetero atoms
- A01N43/647—Triazoles; Hydrogenated triazoles
- A01N43/653—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P15/00—Biocides for specific purposes not provided for in groups A01P1/00 - A01P13/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/18—Ring systems of four or more rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
- C07D249/10—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D249/12—Oxygen or sulfur atoms
Definitions
- the present disclosure relates to compositions, com- pounds, methods and uses.
- Sweet potato (Ipomoea batatas L.) is the 7 th most im- portant food crop in the world, and a subsistence crop in many continents including Latin America, East Africa and China. It is multiplied vegetatively by planting vine-cuttings (i.e. by clon- ing). Plant viral diseases are mainly transmitted by vectors. How- ever, vine-cutting propagation is also likely to transfer viruses to the new clones.
- SPCSV Sweet potato chlorotic stunt virus
- a composition is disclosed.
- composition may comprise a compound represented by any one of formulas I to IV, or a salt thereof:
- R 1 is H, Cl, F, Br, I, methyl, CF 3 , COOH, COO-R 7 , CONHR 7 , acetyl, alkyl, or aryl;
- R 2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF 3 , COO-R 7 , acetyl, alkyl, or aryl;
- R 3 is H, Cl, F, Br, I, methyl, ethyl, CF 3 , COOH, COO-R 7 , acetyl, SO 2 NHR 7 , CONHR 7 , alkyl, or aryl;
- R 4 is Br, H, CF 3 , COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, acetyl, or COO-R 7 ;
- R 5 is H, acetyl, methyl, ethyl, alkyl, or aryl;
- R 6 is acetyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, cy- clohexenyl, phenyl, naphthyl, pyridyl, COOH, COO-R 7 , halogen, meth- oxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, H, or a C 6 to Os aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , -0(C0)R 7 , methyl, ethyl, propyl, isopropyl, n-bu- tyl, sec-butyl, tert-butyl, pentyl,
- R 8 to R 12 are each independently selected from alkyl, methoxy, ethoxy, acetamido, phenyl, H, and halogen;
- R 14 ,and R 17 are each independently selected from H, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H;
- R 15 and R 16 are each independently selected from H, NH 2 , methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H; and wherein each alkyl is optionally substituted;
- R 18 is H or lower alkyl
- R 19 , R 20 , R 22 , R 23 , R 24 , and R 25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy, ethoxy, and NR 37 ;
- R 21 is H, OH, halogen, methyl, lower alkyl, methoxy, eth- oxy, or wherein
- R 26 to R 30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy;
- R 37 is H, methyl, lower alkyl, phenyl, -(CO)CH 2 CH 2 CH 2 CH 2 CH 3 , - (CO)CH 2 CH 2 CH 2 CH 3 , -(CO)CH 2 CH 2 CH 3 , -(CO)CH 2 CH 3 , or -(CO)CH 3 ; and wherein each alkyl is optionally substituted; wherein R 31 is H, methyl, ethyl, or lower alkyl;
- R 32 , R 33 , R 34 , R 35 , and R 36 are each independently selected from H, OH, methoxy, and ethoxy; and wherein each alkyl is optionally substituted.
- Figures 1A to ID show preparation of CSR3 enzymes and characterization of catalytic activity and oligomerization.
- Figure 1A SDS-PAGE of the purified CSR3 and CSR3-A. The gel was stained with Coomassie Brilliant Blue; flow-through (Ft), washing steps 1, 2 (Wl, W2), elution 1-4 (E1-E4), protein ladder (L).
- Figure IB Western blotting for CSR3 and CSR3-A using a rabbit polyclonal antiserum against CSR3.
- Figure 1C Oligomerization of CSR3 was characterized by size-exclusion chromatography coupled with multi- angle light scattering. The calculated molecular mass was 68.93 kDa.
- Figure ID Agarose gel (2%) electrophoresis of the dsRNA substrate (200 bp) incubated at 37°C for 45 min with CSR3, CSR3- A, or no endoribonuclease (Ctl), DNA ladder (L).
- Figures 2A to 2D show an overview (schematic representa- tion) of the FRET-based assay with CSR3 and CSR3-A.
- the labeled siRNA was incubated with CSR3 ( Figure 2A, FRET-absent condition) or with either CSR3-A or no enzyme ( Figure 2B, FRET-present con- dition).
- Figure 2D Agarose gel (2%) electrophoresis of labeled siRNA incubated for 30 min at 37°C with CSR3, CSR3-A, or without any enzyme (Ctl). All reactions contained 15 ⁇ l of 375 nM labeled siRNA.
- L DNA ladder.
- Figures 3A to 3C show a titration assay with CSR3 and labeled siRNA.
- CSR3 two-fold dilution 575 to 36 nM, plus 0 nM control
- labeled siRNA 375 nM
- Three repli- cate plates zOl, z02, z03
- Figure 3C A linear correlation was found between maximal slopes obtained from neighboring cycles (B) and CSR3 concentration.
- Figure 4 illustrates the structures of the compounds with PI > 90% in the primary screen.
- the black stars indicate the top three compounds identified by the dose-response screen of figure 5A to 5C.
- Figures 5A, 5B and 5C show dose-response curves for the top three most potent inhibitors of CSR3 based on DSS values.
- Figures 6A to 6C illustrate the inhibitor validation assay in planta. Sweet potato co-infected with SPCSV and SPFMV were grown in a medium supplemented with a serial concentration of each com- pound (0.1 nM to 100 mM) containing 0.1% of DMSO. In the control condition, co-infected plants were grown on a media supplemented with 0.1% of DMSO. After 28 days of growth, SPCSV and SPFMV viral accumulation was estimated by measuring the relative expression of coat protein of both viruses by RT-qPCR.
- Figure 7 illustrates schematically the workflow of CSR3 inhibitor identification.
- Four main phases were used for identi- fying inhibitors of CSR3: 1) virtual screening using Glide-dock- ing; 2) laboratory screening at the molecular level using FRET- based HTS, followed by MST and SPR; 3) screening and validation in planta using RT-PCR and ChlF imaging; 4) structural analysis of the identified compounds.
- Figures 8A and 8B show homology modeling and virtual screening.
- Figure 8A Amino acid sequence alignment of CSR3 and RNase III enzymes of Escherichia coli (EcR3), Thermotoga maritima (TmR3), and Aquifex aeolicus (AaR3) was done using MAFFT. The active site of RNase III is composed of four amino acids (black arrows) .
- Figure 8B The modeled structure of CSR3, a dimer, was constructed using I-TASSER, based on similar structures (PDB: 1O0W, 2NUG, 1YZ9 E110Q , 1YYW, 2EZ6 D44N , 1YYO, and 1RC7 D44N ).
- CSR3 The two monomers of CSR3 are shown on the left (darker colour) and and on the right (lighter colour).
- Each CSR3 monomer is composed by an endonuclease domain (endoND) and a dsRNA binding domains (dsRBD).
- endoND endonuclease domain
- dsRBD dsRNA binding domains
- the superposed structure of the endoND active sites of CSR3 and AaR3 (PDB 2NUG with 1.7 A resolution) are highlighted in the out- lined box.
- the active site of CSR3 contains four amino acids (40E, 44D, 126N, and 129E) which are represented by tubes, and corre- sponding amino acids of AaR3 are represented by ball-and-sticks.
- FIG. 9 illustrates CSR3 HTS based on a FRET assay.
- Relative fluorescence units (RFU) as a function of detection cycle number shows the difference between the positive (uncleaved) and negative (cleaved) control reactions, measured with an excitation of 485 ⁇ 6 nm and excitation of 520 ⁇ 5 nm during 12 cycles ( ⁇ 17 min total) at 37°C.
- Figures 10A to 10C show hit selection at the molecular level based on CSR3 activity assay and binding affinity assays using MST and SPR.
- Figure 10A Distribution of compounds in FRET- based screening of 6,620 compounds in one concentration. The per- centage of inhibition threshold (30%), used for selecting 109 com- pounds for validations, is represented by a dash line.
- Figure 10B Venn diagram highlighting the 41 compounds selected according to results of dose-response replicates using HTS (rectangle in the intersection of the circles), by considering compounds from FIMM libraries (FIMM1 and FIMM2) and commercial compounds (Comml).
- Fig- ure 10C Venn diagram displaying the 30 compounds (circled in the intersection of the circles) that selected from dose-response and binding affinity assays for further screening in planta. Specifi- cally, 41 compounds from step B (DSS >4), 36 out of 99 from MST (MST), and 36 out of 56 from SPR (SPR).
- Figures 11A to 11F show inhibitor validation in planta.
- Figure 11A Venn diagram highlighting the five compounds selected (circled in the intersection of the circles) by considering results from in vitro laboratory screening (DSS, MST, SPR, Fig. 4C) and effect of compounds on the accumulation of SPFMV (SPFMV ⁇ 0.6) and SPCSV (SPFMV ⁇ 0.6) viruses.
- FIG 11C Representative pictures of co-infected sweet potatoes grown in Sweet potato-Medium supplemented with 50 mM of the five compounds or with 0.1% of DMSO (control), after 28 days.
- Figure 11E Top-view images of co- infected sweetpotato plants treated with either the four compounds or water (Mock) over a month. Photographs were obtained at 41 dpt by RGB imaging or ChlF imaging. False-color images displaying 4>PSII values pixel by pixel were generated using a heat map color scale from dark blue to red ranging from 0.3 to 0.8.
- Figure 11F Effects of compounds on SPFMV accumulation on sweetpotato grown in the soil after 42 dpt.
- Figure 12 shows a hierarchical cluster based on the struc- ture of the five compounds tested in plants.
- the five compounds were clustered into two classes using the methods Tanimoto Coef- ficient, WardLinkage and threshold 0.5 (ChemBioServer). 2D struc- ture of compounds and their IC50 and DSS values from FRET-based HTS, Kd from affinity binding assays (either by MST or SPR), and viral titers in plants are summarized.
- the aphid-transmitted Sweet potato feathery mottle virus (SPFMV, genus Potyvirus, family Potyviridae) and the whitefly- transmitted Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus, family Closteroviridae) have positive-stranded RNA ge- nomes and infect sweet potato (Ipomoea batatas L.) as well as other Ipomoea spp.
- SPFMV alone may cause no symptoms
- SPCSV may cause mild symptoms such as slight stunting and purpling of lower leaves and mild chlorotic mottle in middle leaves.
- the cultivars may possibly develop severe symptoms, which may lead to great economic losses, when coinfected by both SPFMV and SPCSV.
- SPCSV sweet potato virus disease
- SPCSV is thus an agriculturally important pathogen of sweet potato, espe- cially because it may break down resistance to unrelated viruses during a co-infection, significantly increasing disease severity.
- the synergistic effects of SPCSV on other viruses may be due to its interference with RNA silencing because they are often asso- ciated with substantially increased accumulation of coinfecting viruses.
- Class 1 RNase III encoded by SPCSV has been shown to suppress RNA interference (RNAi)-based antiviral defense system in sweet potato.
- the compounds disclosed in this specification may inhibit RNase III encoded by the genomic RNA of SPCSV (CSR3). They may, therefore, be used to treat or prevent SPCSV infections and/or coinfections of SPCSV with one or more other plant pathogenic viruses.
- CSR3 genomic RNA of SPCSV
- composition may comprise a compound represented by any one of formulas I to IV, or a salt thereof:
- R 1 is H, Cl, F, Br, I, methyl, CF 3 , COOH, COO-R 7 , CONHR 7 , acetyl, alkyl, or aryl;
- R 2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF 3 , COO-R 7 , acetyl, alkyl, or aryl;
- R 3 is H, Cl, F, Br, I, methyl, ethyl, CF 3 , COOH, COO-R 7 , acetyl, SO 2 NHR 7 , CONHR 7 , alkyl, or aryl;
- R 4 is Br, H, CF 3 , COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, acetyl, or COO-R 7 ;
- R 5 is H, acetyl, methyl, ethyl, alkyl, or aryl;
- R 6 is acetyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, cy- clohexenyl, phenyl, naphthyl, pyridyl, COOH, COO-R 7 , halogen, meth- oxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, H, or a C 6 to C 8 aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , -0(C0)R 7 , methyl, ethyl, propyl, isopropyl, n-bu- tyl, sec-butyl, tert-butyl, pentyl,
- R 8 to R 12 are each independently selected from alkyl, methoxy, ethoxy, acetamido, phenyl, H, and halogen;
- R 14 ,and R 17 are each independently selected from H, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H;
- R 15 and R 16 are each independently selected from H, NH 2 , methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H; and wherein each alkyl is optionally substituted;
- R 18 is H or lower alkyl
- R 19 ,R 20 ,R 22 ,R 23 ,R 24 , and R 25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy, ethoxy, and NR 37 ; H, halogen, methyl, lower alkyl, methoxy, eth- oxy, or wherein
- R 26 to R 30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy;
- R 37 is H, methyl, lower alkyl, phenyl, -(CO)CH 2 CH 2 CH 2 CH 2 CH 3 , - (CO)CH 2 CH 2 CH 2 CH 3 , -(CO)CH 2 CH 2 CH 3 , -(CO)CH 2 CH 3 , or -(CO)CH 3 ; and wherein each alkyl is optionally substituted;
- R 31 is H, methyl, ethyl, or lower alkyl
- R 32 , R 33 , R 34 , R 35 , and R 36 are each independently selected from H, OH, methoxy, and ethoxy; and wherein each alkyl is optionally substituted.
- composition may comprise a compound represented by any one of formulas I to IV, or a salt thereof:
- Ri is H, Cl, F, Br, I, methyl, CF 3 , COOH, COO-R 7 , alkyl, or aryl;
- R 2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF 3 , COO-R 7 , alkyl, or aryl;
- R 3 is H, Cl, F, Br, I, methyl, ethyl, CF 3 , COOH, COO-R 7 , alkyl, or aryl;
- R 4 is Br, H, CF 3 , COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, or COO-R 7 ;
- R 5 is H, methyl, ethyl, alkyl, or aryl
- R 6 is methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, phenyl, COOH, COO-R 7 , halogen, methoxy, ethoxy, trifluoromethyl, tribro- momethyl, trichloromethyl, H, or a Ce to Ce aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, halogen, methoxy, ethoxy, trifluorome- thyl, tribromomethyl
- R 8 to Ri 2 are each independently selected from alkyl, methoxy, ethoxy, H, and halogen;
- Ri 4 and R 17 are each independently selected from H, methyl, and lower alkyl;
- Ri 5 and Ri 6 are each independently selected from H, N3 ⁇ 4, methyl and lower alkyl; and wherein each alkyl is optionally substituted;
- R 18 is H or lower alkyl
- R 19 ,R 20, R 22, R 23, R 24 , and R 25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy, and ethoxy;
- R 21 is H, OH, halogen, methyl, lower alkyl, methoxy, eth- oxy, or wherein
- R 26 to R 30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy; and wherein each alkyl is optionally substituted; wherein R 31 is H, methyl, ethyl, or lower alkyl;
- R 32 , R 33 , R 34 , R 35 , and R 36 are each independently selected from H, OH, methoxy, and ethoxy; and wherein each alkyl is optionally substituted.
- the composition may be a composition for inhibiting RNase III encoded by SPCSV and/or for reducing the accumulation of at least one plant pathogenic virus in a plant.
- the composition may be a composition for treating a plant curatively and/or preventively against symptoms caused by at least one plant pathogenic virus and/or against sweet potato virus dis- ease (SPVD).
- SPVD sweet potato virus dis- ease
- the composition may be a crop protection composition.
- the composition may further comprise one or more addi- tives, vehicles, formulation auxiliaries, extenders, fillers, and/or surface-active agents.
- the one or more additives, vehicles, formulation auxiliaries, extenders, fillers, and/or surface-active agents may be agriculturally acceptable.
- the composition may, al- ternatively or additionally, comprise at least one pesticidally active substance.
- the at least one pesticidally active substance may comprise or be a pesticidally active substance ef- fective against one or more vectors of plant viruses, such as aphids and/or whiteflies, or other vector(s) capable of spreading any plant pathogenic virus described in this specification.
- the pesticidally active substance may be, for example, an insecticide, acaricide, herbicide, fungicide, safener, or growth regulator.
- a method for treating a plant curatively and/or preven- tively against at least one plant pathogenic virus and/or against sweet potato virus disease is also disclosed.
- the method may comprise applying a compound represented by any one of formulas I to IV according to one or more embodiments described in this specification or a salt thereof, or any compound described in this specification or a salt thereof, or a composition according to one or more embodiments described in this specification, to the plant or a part thereof, a cutting for growing the plant, and/or a tuberous root of the plant, or to soil and/or to substrate in which the plant is growing or in which it is desired to grow.
- a compound, or a composition comprising a compound, is also disclosed, wherein the compound is capable of inhibiting RNase
- SPCSV Sweet potato chlorotic stunt virus
- iden- tified as capable of inhibiting RNase III encoded by SPCSV by a binding assay and/or as capable of reducing the accumulation of at least one plant pathogenic virus in a plant.
- the binding assay may comprise or be an assay (or one or more assays) based on fluorescence resonance energy transfer (FRET), microscale thermophoresis (MST) and/or surface plasmon resonance (SPR).
- FRET fluorescence resonance energy transfer
- MST microscale thermophoresis
- SPR surface plasmon resonance
- the RNase III encoded by Sweet potato chlorotic stunt virus may have an amino acid sequence set forth in the GenBank accession no. GenBank: ADQ42569.1 and/or in SEQ ID NO: 1.
- the compound may be a compound repre- sented by formula I, or a salt thereof:
- Ri is H, Cl, F, Br, I, methyl, CF 3 , COOH, COO-R 7 , CONHR 7 , acetyl, alkyl, or aryl;
- R 2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF 3 , COO-R 7 , acetyl, alkyl, or aryl;
- R 3 is H, Cl, F, Br, I, methyl, ethyl, CF 3 , COOH, COO-R 7 , acetyl, SO 2 NHR 7 , CONHR 7 , alkyl, or aryl;
- R 4 is Br, H, CF 3 , COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, acetyl, or COO-R 7 ;
- R 5 is H, acetyl, methyl, ethyl, alkyl, or aryl;
- R 6 is acetyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, cy- clohexenyl, phenyl, naphthyl, pyridyl, COOH, COO-R 7 , halogen, meth- oxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, H, or a C 6 to Os aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , -0(C0)R 7 , methyl, ethyl, propyl, isopropyl, n-bu- tyl, sec-butyl, tert-butyl, pentyl,
- the compound may be a compound repre- sented by formula I, or a salt thereof:
- R 7 is H, Cl, F, Br, I, methyl, CF 3 , COOH, COO-R 7 , alkyl, or aryl;
- R 2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF 3 , COO-R 7 , alkyl, or aryl;
- R 3 is H, Cl, F, Br, I, methyl, ethyl, CF 3 , COOH, COO-R 7 , alkyl, or aryl;
- R 4 is Br, H, CF 3 , COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, or COO-R 7 ;
- R 5 is H, methyl, ethyl, alkyl, or aryl
- R 6 is methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, phenyl, COOH, COO-R 7 , halogen, methoxy, ethoxy, trifluoromethyl, tribro- momethyl, trichloromethyl, H, or a C 6 to Cs aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclo- pentyl, cyclohexyl, halogen, methoxy, ethoxy, trifluoromethyl, tribromomethyl
- R 1 may, in some embodiments, be H, Cl, F, Br, or I.
- R 1 may, in some embodiments, be H or Cl.
- R 2 may, in some embodiments, be H or COOH.
- R 3 may, in some embodiments, be H, Cl, F, Br, or I.
- R 3 may, in some embodiments, be H or Cl.
- R 4 may, in some embodiments, be Br, H, CF 3 , COOH, Cl, F, I, or methyl.
- R 4 may, in some embodiments, be Br, H, CF 3 , or COOH.
- R 5 may, in some embodiments, be H or methyl.
- R 5 may, in some embodiments, be H.
- Rg may, in some embodiments, be carboxyphenyl, methylphenyl, fluorophenyl, fluorophenyl, phenyl, ethoxycarbonyl, COOH, isopropyl, substituted aromatic ring, alkyl, or COO-R 7 .
- Rg may, in some embodiments, be carboxyphenyl (0-, m-, or p-carboxyphenyl, in particular, p-carboxyphenyl), methylphenyl (o- , m-, or p-methylphenyl, in particular, p-methylphenyl), fluoro- phenyl (0-, m-, or p-fluorophenyl, in particular, p-fluorophenyl or o-fluorophenyl), phenyl, ethoxycarbonyl, COOH, or isopropyl.
- R 7 may, in some embodiments, be alkyl, such as methyl or ethyl.
- the compound may be a compound repre- sented by formula I, or a salt thereof:
- R 3 is H, Cl, F, Br, or I
- R 4 is Br, H, CF 3 , COOH, Cl, F, I, or methyl;
- R 5 is H, or methyl
- R 6 is p-carboxyphenyl, p-methylphenyl, p-fluorophenyl, o- fluorophenyl, phenyl, ethoxycarbonyl, COOH, isopropyl, substituted aromatic ring, alkyl, or COO-R 7 ; wherein each R 7 is independently alkyl or aryl; and wherein each alkyl and/or aryl is optionally substituted.
- SMILES notation has been originally described e.g. in Anderson E, Veith GD, Weininger D (1987). SMILES: A line notation and com- puterized interpreter for chemical structures. Duluth, MN: U.S. EPA, Environmental Research Laboratory-Duluth . Report No. EPA/600/M-87/021.
- Examples of compounds represented by formula I include (but are not limited to) the following:
- the compound may be a compound repre- sented by formula II, or a salt thereof: wherein
- R 8 to R 12 are each independently selected from alkyl, methoxy, ethoxy, acetamido, phenyl, H, and halogen;
- R 14, and R 17 are each independently selected from H, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H;
- R 15 and R 16 are each independently selected from H, NH 2 , methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R 7 , methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H; and wherein each alkyl is optionally substituted.
- the compound may be a compound repre- sented by formula II, or a salt thereof:
- R 8 to R 12 are each independently selected from alkyl, methoxy, ethoxy, H, and halogen;
- R 14 and R 17 are each independently selected from H, methyl, and lower alkyl
- R 15 and R 16 are each independently selected from H, NH 2 , methyl and lower alkyl; and wherein each alkyl is optionally substituted.
- R 8 to R 12 may, in some embodiments, be each independently selected from alkyl, methoxy, H, and halogen.
- R 8 and Rio may, in some embodiments, both be halogen.
- R 8 and R 11 may, in some embodiments, both be lower alkyl.
- R 8 and R 11 may, in some embodiments, both be methyl or ethyl.
- R 8 and R 11 may, in some embodiments, both be methyl.
- R 9 and R 11 may, in some embodiments, both be halogen.
- R 10 and R 11 may, in some embodiments, both be methoxy or ethoxy, or they may be independently selected from H, methoxy and ethoxy.
- R 15 may, in some embodiments, be H.
- R 16 may, in some embodiments, be H or NH 2 .
- Examples of compounds represented by formula II include (but are not limited to) the following:
- Examples of compounds represented by formula II include (but are not limited to) the following:
- the compound may be a compound repre- sented by formula III, or a salt thereof:
- R 18 is H or lower alkyl
- R 19 , R 2 0 , R 22 , R 23 , R 24 , and R 25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy, ethoxy, and
- R 21 is H, OH, halogen, methyl, lower alkyl, methoxy, eth- oxy, or wherein
- R 26 to R 30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy;
- R 37 is H, methyl, lower alkyl, phenyl, -(CO)CH 2 CH 2 CH 2 CH 2 CH 3 , - (CO)CH 2 CH 2 CH 2 CH 3 , -(CO)CH 2 CH 2 CH 3 , -(CO)CH 2 CH 3 , or -(CO)CH 3 ; and wherein each alkyl is optionally substituted.
- the compound may be a compound repre- sented by formula III, or a salt thereof:
- R 18 is H or lower alkyl
- R 19 , R 2 0 , R 22 , R 23 , R 24 , and R 25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy and ethoxy;
- R 21 is H, OH, halogen, methyl, lower alkyl, methoxy, eth- oxy, or wherein
- R 26 to R 30 are each independently selected from H , OH, halogen, lower alkyl, methoxy and ethoxy; and wherein each alkyl is optionally substituted.
- R 18 may, in some embodiments, be H.
- R 19 , R 2 0 , R 22 , R 23 , R 24 , and R 25 may, in some embodiments, be each independently selected from H and OH.
- R 21 may, in some embodiments, be H.
- R 21 may, in some embodiments, be , wherein
- R 26 to R 30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy.
- R 21 may, in some embodiments, be , wherein
- R26 to R30 are each independently selected from H and OH.
- Examples of compounds represented by formula III include (but are not limited to) the following:
- Examples of compounds represented by formula III include (but are not limited to) the following:
- the compound may be a compound repre- sented by formula IV, or a salt thereof: wherein R 31 is H, methyl, ethyl, or lower alkyl; and R 32 , R 33 , R 34 , R 35 , and R 3 6 are each independently selected from H, OH, methoxy, and ethoxy.
- R 31 may, in some embodiments, be H or methyl.
- R 32 , R 33 , R 34 , R 35 , and R 3 6 may, in some embodiments, be each independently selected from H, OH, and methoxy.
- Examples of compounds represented by formula IV include
- Examples of compounds represented by formula IV include (but are not limited to) the following:
- halogen may be understood as referring to F, Cl, Br, and/or I.
- lower alkyl may be understood as referring to an unbranched (i.e. straight chained) or branched C 1 -C 4 alkyl, such as an unbranched or branched C 1 , C 2 , C 3 or C 4 alkyl.
- Examples of lower alkyls include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, and tert-butyl.
- alkyl may be understood as referring to a monovalent moiety obtained or obtain- able by removing a hydrogen atom from a carbon atom of a hydro- carbon compound, which may be aliphatic or alicyclic, and which may be saturated.
- alkyl includes the sub-class cycloalkyl, and the like.
- C 1-12 alkyl means an alkyl moiety having from 1 to 12 carbon atoms.
- aryl may be understood as referring to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms.
- each ring may have from 5 to 8 ring atoms.
- the prefixes e.g. C 3-20 , C 5-8 , etc.
- C 5-6 aryl as used herein means an aryl group having 5 or 6 ring atoms.
- the ring atoms may be all carbon atoms, as in "carboaryl groups".
- carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C 6 ), naphthalene (C 10 ), azulene (C 10 ), anthracene (C 14 ), phenanthrene (C 14 ), naphthacene (Cis), and pyrene (C 16 ) ⁇
- the aryl is phenyl.
- aryl groups which comprise fused rings include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-lH- indene) (C 9 ), indene (C 9 ), isoindene (C 9 ), tetraline (1,2,3,4- tetrahydronaphthalene (C 10 ), acenaphthene (C 12 ), fluorene (C 13 ), phenalene (C 13 ), acephenanthrene (C 15 ), and aceanthrene (C 16 ) ⁇
- indane e.g. 2,3-dihydro-lH- indene
- C 9 indene
- C 9 tetraline (1,2,3,4- tetrahydronaphthalene
- C 10 tetraline (1,2,3,4- tetrahydronaphthalene
- C 12 acenaphthene
- fluorene
- the ring atoms may include one or more heteroatoms, as in "heteroaryl groups".
- heteroaryl groups include, but are not limited to, those derived from:
- N 1 pyrrole (azole) (C 5 ), pyridine (azine) (C 6 );
- N 1 O 1 oxazole (C 5 ), isoxazole (C 5 ), isoxazine (C 6 );
- N 1 S 1 thiazole (C 5 ), isothiazole (C 5 );
- N 2 imidazole (1,3-diazole) (C 5 ), pyrazole (1,2-diazole) (C 5 ), pyridazine (1,2-diazine) (C 6 ), pyrimidine (1,3-diazine) (C 6 ) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C 6 );
- heteroaryls which comprise fused rings, include, but are not limited to:
- C 13 (with 3 fused rings) derived from carbazole (N 1 ), dibenzofuran (O 1 ), dibenzothiophene (S 1 ), carboline (N 2 ), perimidine (N 2 ), pyridoindole (N 2 ); and,
- substituted alkyl and/or “substituted aryl” may be understood as referring to an alkyl or aryl substituted by one or more substituents selected from -COOH, -COO-R 7 , methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, halogen, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, or H.
- Each R 7 may be independently alkyl or aryl, wherein each alkyl and/or aryl may be optionally substi- tuted.
- the compound may be selected from the following, and/or their salts:
- the compound may be selected from the following, and/or their salts.
- the composition may, alternatively or additionally, comprise a compound selected from the following, and/or their salts:
- Examples of compounds represented by formula I include (but are not limited to) the following:
- the at least one plant pathogenic virus may comprise or be at least one of Sweet potato chlorotic stunt virus (SPCSV) or Sweet potato feathery mottle virus (SPFMV).
- SPCSV Sweet potato chlorotic stunt virus
- SPFMV Sweet potato feathery mottle virus
- the at least one plant pathogenic virus may comprise or be SPCSV, optionally synergistic with at least one other plant pathogenic virus.
- the term "syner- gistic with at least one other plant pathogenic virus” may be understood as referring to SPCSV co-infecting the plant with the at least one other plant pathogenic virus.
- the viruses may, at least in some embodiments, synergistically cause symptoms in the plant that may be more severe than symptoms caused by either (or single) plant pathogenic virus alone.
- SPCSV may be synergistic with one or more Potyviruses (SPFMV, SPLV and/or SPMSV), Cucumoviruses (CMV), Ipomoviruses (SPMMV), Carlaviruses (SPCFV and/or C-6 virus), and/or Cave- moviruses (SPVCV and/or SPCV).
- SPFMV Potyviruses
- SPLV SPLV and/or SPMSV
- CMV Cucumoviruses
- SPMMV Ipomoviruses
- SPMMV Carlaviruses
- SPCFV and/or C-6 virus Carlaviruses
- SPVCV and/or SPCV Cave- moviruses
- Examples of Potyviruses with which SPCSV may be syner- gistic include SPFMV, SPLV, i.e. Sweet potato latent virus, and SPMSV, i.e. Sweet potato mild speckling virus.
- Cucumoviruses with which SPCSV may be syner- gistic include CMV, i.e. Cucumber mosaic virus.
- Ipomoviruses with which SPCSV may be syner- gistic include SPMMV, i.e. Sweet potato mild mottle virus.
- Examples of Carlaviruses with which SPCSV may be syner- gistic include SPCFV, i.e. Sweet potato chlorotic fleck virus, and C-6 virus, i.e. sweet potato C6 virus.
- Cavemoviruses with which SPCSV may be syner- gistic include SPVCV, i.e. Sweet potato vein clearing virus, and SPCV, i.e. Sweet potato caulimo-like virus.
- SPCSV may be synergistic with SPFMV and optionally with one or more of other Potyviruses (SPLV and/or SPMSV), Cucumoviruses (CMV), Ipomoviruses (SPMMV), Carlaviruses (SPCFV and/or C-6 vi- rus), and/or Cavemoviruses (SPVCV and/or SPCV).
- SPLV and/or SPMSV Potyviruses
- CMV Cucumoviruses
- SPMMV Ipomoviruses
- SPCFV and/or C-6 vi- rus Carlaviruses
- SPVCV and/or SPCV Cavemoviruses
- the plant may be a plant of Ipomoea spp.
- the plant may be sweet potato (Ipomoea batatas L.).
- Other plants of Ipomoea spp. may include e.g. species used as crops, such as water spinach (I. aquatica), whitestar potato (I. lacu- nosa), Australian bush potato (I. costata); and/or species that are used as ornamental plants, such as heavenly blue morning glory (I. violacea), tropical white morning glory (I. alba), common morning glory (I. purpurea) and other morning glories.
- Other Ipomoea species may also be contemplated.
- the compound represented by any one of formulas I to IV according to one or more embodiments described in this specifica- tion or a salt thereof, or the composition according to one or more embodiments described in this specification, may be applied to the plant or a part thereof, a cutting for growing the plant, and/or a tuberous root of the plant, or to soil and/or to substrate in which the plant is growing or in which it is desired to grow. They may be applied e.g. using various application types described below, such as foliar spray, and/or by dipping or coating a cutting (such as a stem-cutting or vine-cutting). They may, additionally or alternatively, be applied in combination with cryotherapy of shoot tips, e.g. before or after the cryotherapy. It may be con- sidered as a complementary approach to reduce viral load, e.g. before cryopreservation.
- cryotherapy may be understood as referring to a treatment in which a part of the plant, such as a shoot tip, is exposed briefly to liquid nitrogen, so as to reduce the number of plant pathogens, such as plant pathogenic viruses, present in the part of the plant.
- An example of such cryotherapy is described in Wang et al., Annals of Applied Biology 2009, 154(3), 351-363.
- the compound or the composition may be applied e.g. by band application, by basal application, by broadcast application, by directed spray, by foliar spray, by rope-wick or wiper appli- cation, by stem application, by drench/drip application (chemiga- tion), or by injection to the plant.
- the compound or the composition may be applied e.g. by soil application, by soil incorporation, by soil injection, by space treatment, or by spot treatment to soil and/or to substrate in which the plant is growing or in which it is desired to grow.
- the substrate in which the plant is growing or in which it is desired to grow may comprise or be any suitable solid or liquid substrate. Examples of substrates may include pumice, py- roclastic material, synthetic organic substrate, organic sub- strate, and liquid substrate.
- the term "treating a plant curatively against at least one plant pathogenic virus and/or against sweet potato virus disease (SPVD)” may be understood as referring to treating the plant that is infected by the at least one plant pathogenic virus and/or exhibits SPVD.
- the plant may be treated to alleviate the symptoms caused by the at least one plant pathogenic virus and/or by SPVD.
- treating a plant preventively against at least one plant pathogenic virus and/or against sweet potato virus disease may be understood as referring to treating the plant to prevent infection by the at least one plant pathogenic virus and/or to prevent SPVD.
- sweet potato virus disease may be understood as referring to a plant disease caused by co-infection by both SPFMV and SPCSV, for example in a plant of Ipomoea spp., such as in sweet potato.
- CSR3 characteristics for FRET-Based High-Throughput Screening (HTS) for viral RNase III Inhibitors For the development of an HTS assay, His-tagged CSR3 (SEQ ID NO: 2) and its double-mutant CSR3-A (D37A, D44A; SEQ ID NO: 3) were expressed in E. coli and purified with Nickel-nitrilotri- acetic acid (Ni-NTA) agarose.
- Ni-NTA Nickel-nitrilotri- acetic acid
- C-terminal His-tagged CSR3 (GenBank: ADQ42569.1; SEQ ID NO: 2) and CSR3-A (D37A, D44A; SEQ ID NO: 3) were expressed in E. coli from the plasmid pETlld (Kreuze et al., 2005: J Virol, 79, 7227-7238). Bacterial cultures were grown under selection with ampicillin (100 yg/ml) and chloramphenicol (25 yg/ml) at 37°C for 2 h. Recombinant proteins were induced by adding 0.1 mM (final concentration) of isopropyl b-d-l-thiogalactopyranoside (IPTG) into the culture medium and growing for 4 h.
- IPTG isopropyl b-d-l-thiogalactopyranoside
- Bacterial cells were lysed in lysis buffer (10 mM imidazole) supplemented with 1 tablet of protease inhibitor cocktail (complete ULTAR Tablets, mini, Roche, Basel, Switzerland) per 10 ml lysis buffer and 1 mg/ml lysozyme (Sigma-Aldrich, St. Louis, MO, USA) while incubating for 2 h on ice. Sonication (50% duty cycle, 4 c 15 s; Branson Sonifier [B150R Cell Disruptor B15]) was used to additionally disrupt cells and degrade nucleic acids.
- Ni-NTA agarose gravity-flow chromatography (Qiagen, Venlo, Netherlands) was used to purify His-tagged proteins.
- the bacterial extract was loaded onto polypropylene columns (Qiagen) and washed successively with wash buffer 1 (50 mM imidazole) and wash buffer 2 (70 mM imidazole). Bound proteins were eluted with elution buffer (500 mM imidazole). Lysis buffer, wash buffer, and elution buffer were made using the His Buffer kit from GE Healthcare (Chicago, IL, USA).
- Coomassie Bril- liant Blue reagent 10% (v/v) glacial acetic acid, 40% (v/v) meth- anol, 1% (w/v) Coomassie Brilliant Blue G] was used to visualize proteins in gels following SDS-PAGE.
- Western blotting 10% (v/v) glacial acetic acid, 40% (v/v) meth- anol, 1% (w/v) Coomassie Brilliant Blue G
- Proteins were resolved via Tris-glycine SDS-PAGE (12% polyacrylamide) and transferred to polyvinylidene difluoride mem- brane (GE Healthcare) by electroblotting. After SDS-PAGE electrob- lotting, gels were stained with Coomassie Brilliant Blue to confirm that the majority of proteins were transferred.
- PBS phosphate-buffered saline
- PBS-Tween phosphate-buffered saline
- each membrane was incubated for 1 h with a CSR3-specific rabbit poly- clonal antibody (Kreuze et al., 2005: J Virol, 79, 7227-7238) diluted 1:1000 in PBS-Tween containing 2.5% dried milk.
- each membrane was soaked for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma-Aldrich) diluted 1:5000, and bands were visualized with the West Pico chem- iluminescence development substrate (Thermo Fisher Scientific). Chemiluminescence was detected with X-ray film (Roche) using an enhanced chemiluminescence kit from GE Healthcare.
- the oligomerization of CSR3 was also characterized by size-exclusion chromatography with detection using multi-angle light scattering.
- Size-exclusion chromatography coupled with multi-angle light scattering was used for characterizing oligomeric states of CSR3.
- Samples of purified recombinant CSR3 diluted in PBS were loaded onto a Superdex S-20010/300 GL (GE Healthcare) column at 0.5 ml/min with an HPLC system (Shimadzu, Kyoto, Japan) coupled with MiniDAWN TREOS multi-angle light-scattering detector, and Optilab rEX refractive index detector (Wyatt Technology, Santa Barbara, CA, USA).
- the injection volume was 100 ⁇ l per sample, and chromatography was carried out at 4°C.
- the concentration of protein in the effluent was measured with a light-scattering detector (493- TS; RI instrument 686-REX, 658 nm; UV instrument SPD-M20A). Data were analyzed with ASTRA 6 software (Wyatt Technology).
- the CSR3 activity was evaluated using a 200-bp double- stranded RNA (dsRNA) substrate.
- dsRNA double- stranded RNA
- the dsRNA molecules (200 bp) were generated using the TranscriptAid T7 High-Yield Transcription kit (Thermo Fisher Sci- entific).
- To precipitate dsRNA samples were incubated with 2.5 M ammonium acetate on ice for 15 min and centrifuged at 10,000 x g for 15 min at 4°C. After removing the supernatant, the pellet was washed twice with 70% ethanol, air-dried at room temperature for 10 min, and resuspended in RNase-free water (50 ⁇ l). To reanneal RNA as a double strand, samples were incubated at 95°C for 10 min, 65°C for 1 min, and room temperature before storage at -20°C.
- RNase activity was tested in 20 ⁇ l reactions (20 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2,pH 8) containing 300 ng of 200-bp dsRNA and 200 ng enzyme. Each reaction was incubated for 40 min at 37°C before loading on a 1% agarose gel.
- siRNA small interfering RNA constructs A 22 bp 2-nt 3'-overhang siRNA labeled with a FAM reporter and BHQ1 quencher (forward: 5' FAM-CGUAGUGGAAGUGGGAGAGGTC-BHQ13' [unlabeled sequence set forth in SEQ ID NO: 4]; reverse: 5' CCU- CUCCCACUUCCACUACGTG 3' [SEQ ID NO: 5]) were synthesized by Metabion (Munich, Germany). Their identity and purity were verified by HPLC.
- RNA oligonucleotides were dissolved in annealing buffer (6 mM HEPES pH 7.5, containing 60 mM KC1 and 0.2 mM MgCl2) at a concen- tration of 100 mM, aliquoted in volumes ranging from 10 to 200 ⁇ l and stored at -20°C. Before use, siRNA was diluted to 15 mM (200 ng/ ⁇ l) with annealing buffer, incubated 2 min at 93°C and cooled to room temperature (330 min).
- annealing buffer 6 mM HEPES pH 7.5, containing 60 mM KC1 and 0.2 mM MgCl2
- the HTS assay was designed to maximize siRNA cleavage by CSR3.
- a donor-quencher fluorogenic siRNA was used as the substrate for CSR3.
- a CSR3 titration assay was carried out by mixing six different concen- trations of CSR3 (0-1150 nM) with 375 nM labeled siRNA according to results obtained from a preliminary test.
- full plates containing only negative (50 nM CSR3) and positive reactions (lacking CSR3) were tested with 375 nM labeled siRNA.
- Enzyme and substrate were pre- pared separately at 2x final concentration, and then 10 ⁇ l of each was dispensed into every well with an automated dispenser (MultiFlo FX with single-channel RAD-cassettes; BioTek, Winooski, VT, USA). Plates were 384-well black flat-bottom microplates (#3544, Corn- ing, NY, USA).
- the HTS assay was based on FRET, in which CSR3 cleaves a labeled siRNA and generates a fluorescent signal (Fig. 2A, B).
- the 2-nt overhang of a 22-bp siRNA labeled with a fluor- ophore reporter (FAM) and a quencher (HBQ1) at the 5' and 3' end, respectively, of the sense-strand was used as substrate. Fluores- cence was acquired with excitation/emission of 485/520 nm, re- flecting the unquenched fluorescence intensity of the reporter. Two reaction conditions were used for the negative and positive controls to normalize CSR3-inhibition data acquired for all com- pounds. For the negative control, cleavage of the labeled siRNA by CSR3 disrupts the energy transfer from the donor to the receptor and hence loss of FRET quenching, allowing the detection of the fluorescence emission of the reporter (Fig.
- the endoribonuclease activity of CSR3 was calculated with slope, representing fluorescence changes in function of assay time (s), using MARS Data Analysis software (BMG Labtech).
- the Z' of the assay was calculated according to Eq. A (Zhang et al., 1999, J Biomol Screen, 4, 67-73), which was used to evaluate the suitability of the method during assay development, optimiza- tion, and screening.
- signal-to-noise ratio ( ⁇ c _ - ⁇ c+ ) / o c+
- signal-to-background ratio ⁇ c _ / ⁇ c+ .
- the percentage of inhibition (PI) of each compound for CSR3 was calculated according to Eq. B.
- a PI threshold of 30% was used as the cutoff value for one concentration HTS.
- the dose-response curves of PI in function of compounds' concentra- tion were evaluated with DSS according to Yadav et al. (2014),
- the kinetic constant (Kd) for CSR3 was calculated using the three-parameter Michaelis-Menten model (MM.3) included in the R package dcr (Ritz et al., 2015, PLoS One, 10, e0146021). The statistical significance of differences between values was as- sessed with one-way ANOVA using the aov function in the R package.
- the rate of increase in fluorescence was dependent on the amount of enzyme and labeled siRNA in the reaction. Fluorescence measurements (Fig. 3A) showed that, compared with a low concen- tration, a high concentration of CSR3 led to a rapid increase and higher fluorescence at the beginning of the kinetic measurement.
- the slopes were calculated between all neighboring detection cycles. The slopes were used to select the optimal detection time for each CSR3 concentration tested. The results suggested that the maximal initial slope for the concentrations 144 nM, 72 nM, and 36 nM of CSR3 occurred after 5, 10, and 14 reaction cycles, respectively (Fig.
- Table 1 Calculated Z' values for the five different CSR3 concentrations used in the three replicates over 12 cycles.
- the final CSR3 and substrate concentrations were 100 nM and 375 nM, respec- tively.
- the dose-response assay with six compound concentrations was carried out with the same conditions. Plates were sealed, centrifuged briefly, and then immediately an- alyzed with a PHERAstar FS (BMG Labtech, Ortenberg, Germany) with a fluorescence-intensity optic module (excitation at 485 ⁇ 6 nm, detection at 520 ⁇ 5 nm) for 12 cycles ( ⁇ 17 min total) at 37°C. All dispensing was done using the BioTek MultiFlo FX.
- All screening plates contained positive-control wells (lacking CSR3) and nega- tive-control wells (25 nl dimethyl sulfoxide, vehicle), which were used as standards to calculate the percentage of inhibition (PI) of each compound (see equation above in Data analysis).
- the FRET-based assay was utilized in a primary screen of 6620 small molecules of diverse structure. Of these 6620 compounds, 109 (1.66%) had a PI >30% (for PI distribution of those 109 com- pounds, see Table 2). The 12 compounds with a PI value >90% had diverse structures, and no common scaffold was readily apparent (Fig. 4).
- a dose-response assay was carried out with the top 109 compounds (concentration range, 1.25 nM to 50 ⁇ M). The data were analyzed with Breeze software, which generates dose-response curves and calculates the half-maximal inhibitory concentration (IC50) and drug-sensitivity score (DSS) for every compound. Con- sidering that IC50 alone cannot comprehensively evaluate drug sen- sitivity of dose-response model in HTS assay, DSS was developed by Yadav et al. (2014), Sci Rep, 4, 5193 as a systematic algorithmic solution that integrated five factors including IC50, the slope at IC50, minimum activity level, and top and bottom asymptotes of dose-response model.
- IC50 half-maximal inhibitory concentration
- DSS drug-sensitivity score
- the DSS values ranged from 0 to 19.2.
- Table 3 and Figs. 5A to 5C present data for the top 3 compounds based on DSS values. The three structures differ (Fig. 4, starred com- pounds).
- 10 were among the top 20 most potent compounds as determined in the dose-response screen, indicating that the assay was internally consistent even though a single CSR3 concentration was used.
- Table 3 The top three most potent inhibitors of CSR3 as determined with the dose-response assay based on DSS values.
- CSR3 inhibitors were identified in the assays with a PI value of 30% as a threshold. By raising the threshold, one is able to effectively reduce the number of poten- tial false positives, and induce false negatives.
- FIMM031755 (CID: 7114450) has been involved in an inhibitor screening assay for Cytokine/receptor Binary Complex of humans (4KC3_B) and was shown to be effective at micromolar concentra- tions.
- FIMM027745 (CID: 712810) has been used in several inhibitor screens, e.g. Hiv-1 Reverse Transcriptase and human heat shock 70 kDa protein, but was inactive in all studies.
- the HTS assay could be used to identify inhibitors of various class 1 RNase III enzymes.
- Inhibitor identification for CSR3 was done in four phases (Fig. 7).
- Phase one CSR3 structure was modeled and virtual screening by Glide-docking was performed with 136,353 compounds targeting the active site of CSR3.
- phase two compound screening in laboratory was performed including a FRET-based HTS set up and two binding affinity assays using MST and SPR.
- Phase three was a validation assay in planta where the effects of inhibitors on viral accumulation were monitored using RT-qPCR and imaging-based meth- ods in sweet potato grown in medium or soil, respectively.
- Phase four was a posterior cluster study of the hits based on the com- pound structures.
- CSR3 was rather similar in size to RNase III from E.coli (EcR3), Aquifex aeolicus (AaR3) and Thermotoga maritima (TmR3) with 228, 226, 221 and 240 residues, respectively (Fig. 8A). All these proteins are proto- typical class 1 RNase III enzymes which have been well studied structurally and functionally. These enzymes are composed of an endoND and a dsRBD connected by a flexible linker as represented by the CSR3 structure in Fig. 8B. The catalytic site is composed of four amino acids 40E, 44D, 126N, and 129E in CSR3 (Fig.
- 107D in AaR3 corresponds to 126N in CSR3, see the superposed catalytic site of CSR3 and AaR3 (PDB 2NUG, 1.7 A) in Fig. 8B.
- These amino acids are essential for the catalytic activity of the enzyme.
- the side chains of these four amino acids are negatively charged or can be deprotonated allowing attraction of positively charged metals e.g. Mg 2+ , which further attract the negatively charged phosphates (-P0 4- ) of dsRNA.
- the two-metal-ion mediated catalytic mechanism of RNase III has been described earlier.
- 136,353 compounds were Glide-docked into the catalytic site of CSR3, and 6,620 compounds were selected for further experimental testing according to their GlideScore rank order.
- the model of CSR3 was built by I-TASSER.
- I- TASSER identify templates structure by LOMETS server, then select and score the templates of the highest significance in the thread- ing alignments which were used to simulate a pool of protein structure decoys.
- the top five models are identified ac- cording to pair-wise structure similarity using SPICKER program (Roy et al., 2010, Nature Protocols 5(4):725-738).
- the top identified template structures are PDB 1O0W, 5B16, 3C4T,
- the highest-ranked model with I-TASSER c-score 0.56, TM-score 0.7910.09 was selected to further Glide-docking.
- the selected CSR3 model was processed with Protein Preparation Wizard of Schrodinger (Schrodinger releases 2016-4: LLC, New York, NY). Structures of 136,353 small molecules from the High Throughput Biomedicine Unit (HTB) of the Institute for Molecular Medicine Finland (FIMM) were prepared with LigPreg function of Schrodinger with default setup.
- HTB High Throughput Biomedicine Unit
- FIMM High Throughput Biomedicine Unit
- Active site residues of CSR3 (40E, 44D, 126N, and 129E) were selected as the center of Glide-Grid box, and docking was performed using SP and XP scoring modes employing OPLS3 force field under default settings (Friesner et al., 2004, J Med Chem 47(7):1739-1749; Friesner et al., 2006, J Med Chem 49(21):6177-6196). Based on ranking of GlideScore, 6,620 out of 136,353 compounds were selected to laboratory screening.
- 6,620 compounds selected from Glide-docking were screened using the HTS assay described in the Examples above.
- dose-response curve for each compound was generated using the PI values, and then half-maximal inhibitory concentration (IC50) and drug sensitivity score (DSS) were deter- mined according to Yadav et al. (2014), Sci Rep, 4, 5193. The DSS value was used to score the sensitivity of individual compounds.
- the results showed that DSS values varied between 0 and 22, taking threshold DSS >4, 41 compounds were selected for the next step in consideration of all three replicates in Fig. 10B (rectangle in the intersection of the circles).
- Proteins were labeled using Red-Tris-NTA dye (NanoTemper, Miinchen, Germany) and resuspended in 50 ⁇ l of PBS buffer (137 mM NaCl, 2.7 mM KC1, 10 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 , pH 7.4) with 0.05% Tween-20 to obtain 5 mM dye solution.
- Labelled-protein solution containing 500 nM proteins and 40 nM dye was prepared in PBS buffer with 2% DMSO for the assay. The 12 concentrations for each compound were obtained by 2-fold serial dilutions (400 mM — 2 mM).
- a peptide control was performed to discriminate binding-specific fluores- cence quenching from loss of fluorescence due to protein precipi- tation.
- Two independent experiments were carried out in Premium Coated Capillaries using MST power set on high (80%); LED power (pico red) set on 5% and on-Time 20 s.
- the dissociation constant (Kd) was determined using the MO.Affinity Analysis (NanoTemper).
- Binding affinity assay was carried out with the 99 com-fural compounds. Firstly, all 99 compounds were tested using MST. As a result, 36 compounds were selected as binders by taking into consideration four conditions: 1) raw fluorescence induced by compounds excluding their initial fluorescence, 2) signal/noise >5, 3) response amplitude >4, and 4) Kd lower than 200 mM (Fig 10C, the top right circle indicated as "MST").
- Sweet potato (cultivar Huachano, CIP42006) were side graft-inoculated with both SPFMV (East African strain isolate Naml) and SPCSV-Ug (East African serotype 2) as described in Buche et al., 2008, Mol Plant Pathol 9(5):589-598 and Wang et al., 2008, J Virol Methods 154(1-2):135-145. Plantlets were propagated by taking single-node stem grown in culture medium (Wang et al., 2019, Plant Methods 15(1):116).
- plantlets with newly formed roots were transferred to glass tubes (18 x 150 mm) containing 10 ml of medium supplemented with either 50 mM of compound (diluted in DMSO), or only 0.1% DMSO as control.
- plantlets were transferred to pots (6 x 6 x 10 cm) filled with a mix out of 1/3 sand, 1/3 humus, and 1/3 washed soil.
- plants were treated by foliar spraying using either 10 mM compound (treatment) or water (control), twice a week over a month. All plants were grown at 22°C with 60% of humidity and a 16/8h light/dark photoperiod for 28 days in culture medium and 41 days in soil.
- First-strand cDNA was synthesized using the Transcriptor 1st cDNA synthesis kit (Roche, Basel, Switzerland). Gene expres- sion was measured in a final 10 ⁇ l volume (containing 2 ⁇ l lOx diluted cDNA, 5 ⁇ l SYBR Green I Master Mix (Roche), and 2.5 mM primers) using the LightCycler 480 instrument II (Roche). All RT- qPCR experiments were conducted in triplicate on 3 biological rep- licates. Primer list can be found in Wang et al., 2019, Plant Methods 15(1):116. Relative gene expression was calculated using the classical 2 ⁇ DD0T method since the efficiency of all primer pairs were close to 100% and showed less than 5% difference between them.
- Sweet potato plants co-infected with SPCSV and SPFMV grown on culture medium separately supplemented with 50 mM each compound were used to evaluate the effects of compounds on plants.
- 55 compounds were screened in planta considering the results of HTS and affinity assays. Their effects were monitored by plant height by imaging the plants once a week. 7 of 55 compounds showed stress symptoms, e.g. deformation, wilting, bleaching, dried leaf margins, or severe growth defects, possibly because of their toxicity to plants.
- virus accumulation was quan- tified using the relative expression of their coat proteins as described in Kokkinos & Clark, 2006, Plant Dis. 90(6):783-788 and Buche et al., 2008, Mol Plant Pathol 9(5):589-598, and compounds' effects on accumulation was estimated by comparing treated and control plants.
- SPFMV was reduced two times by three compounds (FIMM022230, FIMM005536, and FIMM051696) and one time by two compounds (FIMM000096 and FIMM031755). Overall, the five compounds reduced both SPCSV and SPFMV accumulation without any phytotoxicity effects in sweet po- tato plants, see experimental plant pictures in Fig. 11C.
- sweet potato plants treated with all four compounds displayed a signif- icant increase of 4>PSII values compared to controls reflecting improved photosynthetic performance (Fig. 11D, Dunnett's test).
- the effects on 4>PSII were confirmed by a significant reduction of SPFMV accumulation (Fig. 11F, Dunnett's test).
- Fig. 11E Dunnett's test
- antiviral strategies in plants are based on either breeding virus-resistant cultivars or targeting viruses to prevent viral replication and spreading.
- Another effective strat- egy widely used for animal viruses and often forsaken in plants, is antiviral drug identification by targeting different stages of the viral life-cycle.
- Most virus control strate- gies are applied in pre-infected plants, which emphasizes the need for the development of alternative anti-viral strategies in post- infected plants such as antiviral inhibitor identification.
- HTS of small molecules may be used for antiviral inhibitor discovery.
- HTS targeting viral RNA silencing suppressors has become a potential approach to control virus diseases.
- RNA silencing suppressor CSR3 encoded by SPCSV.
- Many inhibitors of RNase H have been found, e.g. N-hydroxyimide (2-hydroxy-4H- isoquinoline-1,3dione), F3284-8495, and a series of N-hydroxyimide compounds.
- RNase H enzymes are functionally very differ- ent from class 1 RNase Ills, as RNase H enzymes hydrolyze the RNA strands of DNA/RNA duplexes during reverse transcription. As ex- pected, inhibitors of RNase H were not docking well to CSR3 in silico.
- Glide-docking used in the present examples, is a com- plete and hybrid method for searching potential docking poses with high accuracy.
- CSR3 structural model of CSR3 was used in silico, a relatively large number (6,620) of small mole- cules were screened in laboratory.
- targeting of highly conserved amino acid residues in the active site of CSR3 could reduce resistance breaking, which is an important feature in the development of sustainable antiviral strategies.
- Primary labora- tory screening was carried out using a FRET-based HTS built-up in the lab.
- FRET-based methods have pros, such as sensitivity and efficiency, but also cons which are likely to produce false-posi- tive and false-negative results.
- false-positive results could be obtained in two conditions: 1) compounds could directly interact with the substrate instead of CSR3 to prevent labelled-siRNA cleavage; 2) compounds could exhibit in- trinsic fluorescence with similar absorption and emission spectra as the fluorophore reporter. On the other hand, false-negative findings would be obtained if compounds quench the reporter fluor- ophore.
- MST Two complementary methods, MST and SPR, were used to di- rectly measure the binding affinity between the CSR3 and compounds in our study.
- MST records the motion of molecules in microscopic temperature gradients detecting changes in hydration shell, charge, or size.
- MST is a fluorescence-based method, and thus susceptible to disturbance by intrinsically fluorescent compounds, self-fluorescent compounds with similar fluorescent properties cannot be analyzed by this approach.
- the interaction between protein and compounds is measured by monitoring small changes of an optical reflective index at the sensor surface.
- some compounds cannot properly dissociate from the sensor, which will affect the assay of the next analyte. Thus, such compounds were identified in the prelim- inary one concentration test in SPR and excluded from further dose- response tests.
- class 1 compound FIMM000096 has been approved as a powerful emetic and also been used in the treatment of par- kinsonism, but with adverse effects https://www.drug- bank.ca/drugs/DB00714 .
- the other four compounds, belonging to class 2 appear to have not been reported either in the Drugbank database or for the treatment of virus diseases. But they do have been included in inhibitor screening for human enzymes or bacterial proteins according to PubChem database with FIMM022230 (CID: 2948389), FIMM031755 (CID: 7114450), FIMM005536 (CID: 2857906), FIMM051696 (CID: 4240943).
- Fig. 12 also summarizes results from the FRET-based HTS, binding affinity as- says (MST or SPR) and in planta validation results from sweetpota- toes grown in culture medium.
- FRET-based HTS the compounds had similar DSS and IC50 values ranging from 12.4 to 15.9, and 1.27 to 2.9 mM, respectively. Their diminish of viruses accumula- tion in plants ranging from -0.77 to -3.56.
- the Kd values from binding affinity experiments varied between 0.69 mM to 3.44 mM among these compounds.
- the hit rate was 4.86% in Glide-docking in silico, 1.69% in FRET-based HTS, 36.4% in MST, 75% in SPR and 18.2% in viral screening in plants. Considering all screening steps, four compounds (hit rate 0.0037%) were identified as in- hibitors of CSR3.
- the embodiments described hereinbefore may be used in any combination with each other. Several of the embodiments may be combined together to form a further embodiment.
- a method, a product, or a use, disclosed herein may comprise at least one of the embodiments described hereinbefore. It will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments. The embodiments are not limited to those that solve any or all of the stated problems or those that have any or all of the stated benefits and advantages. It will further be understood that reference to 'an' item refers to one or more of those items.
- the term "comprising" is used in this specification to mean including the feature(s) or act(s) followed thereafter, without excluding the presence of one or more additional features or acts.
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Abstract
A composition comprising a compound or a salt thereof is disclosed. A method for treating a plant curatively and/or preventively against at least one plant pathogenic virus and/or against sweet potato virus disease (SPVD), as well as uses of a or a salt thereof, or of the composition, are also disclosed.
Description
COMPOUNDS AND COMPOSITIONS FOR TREATING SWEET POTATO AGAINST SWEET POTATO PATHOGENIC VIRUSES
TECHNICAL FIELD
The present disclosure relates to compositions, com- pounds, methods and uses.
BACKGROUND
Sweet potato (Ipomoea batatas L.) is the 7th most im- portant food crop in the world, and a subsistence crop in many continents including Latin America, East Africa and China. It is multiplied vegetatively by planting vine-cuttings (i.e. by clon- ing). Plant viral diseases are mainly transmitted by vectors. How- ever, vine-cutting propagation is also likely to transfer viruses to the new clones.
The most harmful pathogens of sweet potato are viruses. Currently, virus-resistant sweet potato cultivars are not availa- ble, and chemical control of virus vectors in sweet potato fields may not be effective. Yield losses are caused by viruses in this crop, in particular by Sweet potato chlorotic stunt virus (SPCSV). It may trigger disease symptoms alone, or worse, its co-infections with many other viruses (synergism) can cause almost total inhi- bition of sweet potato plant growth. SPCSV encodes a class 1 RNase III, which has the ability to suppress the RNA interference (RNAi)- based antiviral defense system in sweet potato.
SUMMARY
This Summary is provided to introduce a selection of con- cepts in a simplified form that is further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
A composition is disclosed.
The composition may comprise a compound represented by any one of formulas I to IV, or a salt thereof:
Formula I wherein R1 is H, Cl, F, Br, I, methyl, CF3, COOH, COO-R7, CONHR7, acetyl, alkyl, or aryl;
R2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF3, COO-R7, acetyl, alkyl, or aryl;
R3 is H, Cl, F, Br, I, methyl, ethyl, CF3, COOH, COO-R7, acetyl, SO2NHR7, CONHR7, alkyl, or aryl;
R4 is Br, H, CF3, COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, acetyl, or COO-R7;
R5 is H, acetyl, methyl, ethyl, alkyl, or aryl;
R6 is acetyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, cy- clohexenyl, phenyl, naphthyl, pyridyl, COOH, COO-R7, halogen, meth- oxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, H, or a C6 to Os aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, -0(C0)R7, methyl, ethyl, propyl, isopropyl, n-bu- tyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclo- hexyl, halogen, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, hydroxyl, nitro, or H; wherein each R7 is independently -CH2CH2CH2COOH, CH2CH2COOH, -CH2COOH, alkyl, aryl, or a C6 to C8 aromatic ring optionally substituted with one or more substituents selected in- dependently from the group consisting of -COOH, -CH2-COOH, -CH2- CH2-COOH, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichlorome- thyl, hydroxyl, nitro, or H; and wherein each alkyl and/or aryl is optionally substituted;
R8 to R12 are each independently selected from alkyl, methoxy, ethoxy, acetamido, phenyl, H, and halogen;
R14,and R17 are each independently selected from H, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H;
R15 and R16 are each independently selected from H, NH2, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H; and wherein each alkyl is optionally substituted;
Formula III wherein
R18 is H or lower alkyl;
R19, R20, R22, R23, R24 , and R25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy, ethoxy, and NR37 ;
R26 to R30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy;
R37 is H, methyl, lower alkyl, phenyl, -(CO)CH2CH2CH2CH2CH3, - (CO)CH2CH2CH2CH3, -(CO)CH2CH2CH3, -(CO)CH2CH3, or -(CO)CH3; and wherein each alkyl is optionally substituted;
wherein R31 is H, methyl, ethyl, or lower alkyl;
R32 , R33 , R34 , R35 , and R36 are each independently selected from H, OH, methoxy, and ethoxy; and wherein each alkyl is optionally substituted.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are included to provide a further understanding of the disclosure and constitute a part of this specification, illustrate embodiments and together with the description help to explain the principles of the disclosure. In the drawings:
Figures 1A to ID show preparation of CSR3 enzymes and characterization of catalytic activity and oligomerization. Figure 1A: SDS-PAGE of the purified CSR3 and CSR3-A. The gel was stained with Coomassie Brilliant Blue; flow-through (Ft), washing steps 1,
2 (Wl, W2), elution 1-4 (E1-E4), protein ladder (L). Figure IB: Western blotting for CSR3 and CSR3-A using a rabbit polyclonal antiserum against CSR3. Figure 1C: Oligomerization of CSR3 was characterized by size-exclusion chromatography coupled with multi- angle light scattering. The calculated molecular mass was 68.93 kDa. Figure ID: Agarose gel (2%) electrophoresis of the dsRNA substrate (200 bp) incubated at 37°C for 45 min with CSR3, CSR3- A, or no endoribonuclease (Ctl), DNA ladder (L).
Figures 2A to 2D show an overview (schematic representa- tion) of the FRET-based assay with CSR3 and CSR3-A. The labeled siRNA was incubated with CSR3 (Figure 2A, FRET-absent condition) or with either CSR3-A or no enzyme (Figure 2B, FRET-present con- dition). Figure 2C: A representation of fluorescence signal curve of the FRET-absent and FRET-present conditions, indicating by real-time relative fluorescence units (RFU) in function of ~17 min total (12 cycles), measured at 37°C by an optic module with exci- tation at 485 ± 6 nm and detection at 520 ± 5 nm. Data represent the mean ± SD (n = 24). Figure 2D: Agarose gel (2%) electrophoresis of labeled siRNA incubated for 30 min at 37°C with CSR3, CSR3-A, or without any enzyme (Ctl). All reactions contained 15 μl of 375 nM labeled siRNA. L, DNA ladder.
Figures 3A to 3C show a titration assay with CSR3 and labeled siRNA. CSR3 (two-fold dilution 575 to 36 nM, plus 0 nM control) and labeled siRNA (375 nM) were used to determine the optimal ratio of enzyme-to-substrate concentrations. Three repli- cate plates (zOl, z02, z03) containing labeled siRNA were prepared with a dispenser, and enzyme was added to initiate the reaction. Figure 3A: Raw fluorescence expressed in RFU as a function of detection cycle. In total, 25 cycles were acquired (38 min total). Data represent the mean ± SD (n = 48). Figure 3B: Slope between neighboring cycles obtained from raw fluorescence data (A). The highest CSR3 concentration (575 nM) was excluded. Data represent the mean ± SD (n = 48). Figure 3C: A linear correlation was found between maximal slopes obtained from neighboring cycles (B) and CSR3 concentration.
Figure 4 illustrates the structures of the compounds with PI > 90% in the primary screen. The black stars indicate the top
three compounds identified by the dose-response screen of figure 5A to 5C.
Figures 5A, 5B and 5C show dose-response curves for the top three most potent inhibitors of CSR3 based on DSS values. Figures 6A to 6C illustrate the inhibitor validation assay in planta. Sweet potato co-infected with SPCSV and SPFMV were grown in a medium supplemented with a serial concentration of each com- pound (0.1 nM to 100 mM) containing 0.1% of DMSO. In the control condition, co-infected plants were grown on a media supplemented with 0.1% of DMSO. After 28 days of growth, SPCSV and SPFMV viral accumulation was estimated by measuring the relative expression of coat protein of both viruses by RT-qPCR. Methods of RNA isolation and RT-qPCR are described in Wang et al., 2019, Plant Methods 15(1):116 . Down-regulation of SPFMV (Figure 6A) and SPCSV (Figure 6B) accumulation induced by each compound relative to control plants, which was represented by log2 fold change of their respec- tive coat proteins expression. Values are mean ± SE (n = 2-3). Figure 6C: Plant images of co-infected sweet potatoes grown in medium supplemented with compounds or with 0.1% of DMSO (control), after 28 days.
Figure 7 illustrates schematically the workflow of CSR3 inhibitor identification. Four main phases were used for identi- fying inhibitors of CSR3: 1) virtual screening using Glide-dock- ing; 2) laboratory screening at the molecular level using FRET- based HTS, followed by MST and SPR; 3) screening and validation in planta using RT-PCR and ChlF imaging; 4) structural analysis of the identified compounds.
Figures 8A and 8B show homology modeling and virtual screening. Figure 8A: Amino acid sequence alignment of CSR3 and RNase III enzymes of Escherichia coli (EcR3), Thermotoga maritima (TmR3), and Aquifex aeolicus (AaR3) was done using MAFFT. The active site of RNase III is composed of four amino acids (black arrows) . Figure 8B: The modeled structure of CSR3, a dimer, was constructed using I-TASSER, based on similar structures (PDB: 1O0W, 2NUG, 1YZ9E110Q, 1YYW, 2EZ6D44N, 1YYO, and 1RC7D44N). The two monomers of CSR3 are shown on the left (darker colour) and and on the right (lighter colour). Each CSR3 monomer is composed by an endonuclease domain (endoND) and a dsRNA binding domains (dsRBD).
The superposed structure of the endoND active sites of CSR3 and AaR3 (PDB 2NUG with 1.7 A resolution) are highlighted in the out- lined box. The active site of CSR3 contains four amino acids (40E, 44D, 126N, and 129E) which are represented by tubes, and corre- sponding amino acids of AaR3 are represented by ball-and-sticks.
Figure 9 illustrates CSR3 HTS based on a FRET assay. Relative fluorescence units (RFU) as a function of detection cycle number shows the difference between the positive (uncleaved) and negative (cleaved) control reactions, measured with an excitation of 485 ± 6 nm and excitation of 520 ± 5 nm during 12 cycles (~ 17 min total) at 37°C. Negative and positive control data are shown as the mean ± SD (n = 240) from the 20 screening plates.
Figures 10A to 10C show hit selection at the molecular level based on CSR3 activity assay and binding affinity assays using MST and SPR. Figure 10A: Distribution of compounds in FRET- based screening of 6,620 compounds in one concentration. The per- centage of inhibition threshold (30%), used for selecting 109 com- pounds for validations, is represented by a dash line. Figure 10B: Venn diagram highlighting the 41 compounds selected according to results of dose-response replicates using HTS (rectangle in the intersection of the circles), by considering compounds from FIMM libraries (FIMM1 and FIMM2) and commercial compounds (Comml). Fig- ure 10C: Venn diagram displaying the 30 compounds (circled in the intersection of the circles) that selected from dose-response and binding affinity assays for further screening in planta. Specifi- cally, 41 compounds from step B (DSS >4), 36 out of 99 from MST (MST), and 36 out of 56 from SPR (SPR).
Figures 11A to 11F show inhibitor validation in planta. Figure 11A: Venn diagram highlighting the five compounds selected (circled in the intersection of the circles) by considering results from in vitro laboratory screening (DSS, MST, SPR, Fig. 4C) and effect of compounds on the accumulation of SPFMV (SPFMV < 0.6) and SPCSV (SPFMV < 0.6) viruses. Figure 11B: Down-regulation of SPCSV and SPFMV accumulation induced by the five compounds in co-infected sweet potato grown in vitro, represented by log2 fold change of the expression of their respective coat proteins, values are mean ± SE (n = 3). Figure 11C: Representative pictures of co-infected sweet potatoes grown in Sweet potato-Medium supplemented with 50
mM of the five compounds or with 0.1% of DMSO (control), after 28 days. Figure 11D: Overall quantum yield of PSII (4>PSII) values of co-infected sweet potatoes treated twice a week with either the four compounds or water (Ctl_l and Ctl_2) over a month. Experiments were carried out in two independent batches, each one including their mock condition and n = 30 and 35, respectively. Values of 4>PSII (black point) measured from 37 to 41 days post transfer (dpt) are shown in a boxplot. Significant annotations indicating 0 '***' 0.001 '**' 0.01 0.05 by Dunnett's test for comparing several treatments with a control. Figure 11E: Top-view images of co- infected sweetpotato plants treated with either the four compounds or water (Mock) over a month. Photographs were obtained at 41 dpt by RGB imaging or ChlF imaging. False-color images displaying 4>PSII values pixel by pixel were generated using a heat map color scale from dark blue to red ranging from 0.3 to 0.8. Figure 11F: Effects of compounds on SPFMV accumulation on sweetpotato grown in the soil after 42 dpt. Compounds' effects on viral accumulation were assessed by fold change of coat protein expression of SPFMV in co- infected sweet potato plants. Data represent the mean ± 95% con- fidence interval, N = 10. Significant annotations indicating 0 '***' 0.001 '**' 0.01 0.05 by Dunnett's test for comparing several treatments with a control.
Figure 12 shows a hierarchical cluster based on the struc- ture of the five compounds tested in plants. The five compounds were clustered into two classes using the methods Tanimoto Coef- ficient, WardLinkage and threshold 0.5 (ChemBioServer). 2D struc- ture of compounds and their IC50 and DSS values from FRET-based HTS, Kd from affinity binding assays (either by MST or SPR), and viral titers in plants are summarized.
DETAILED DESCRIPTION
Compounds and compositions are disclosed.
The aphid-transmitted Sweet potato feathery mottle virus (SPFMV, genus Potyvirus, family Potyviridae) and the whitefly- transmitted Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus, family Closteroviridae) have positive-stranded RNA ge- nomes and infect sweet potato (Ipomoea batatas L.) as well as other Ipomoea spp. At least in some cultivars, SPFMV alone may cause no
symptoms, whereas SPCSV may cause mild symptoms such as slight stunting and purpling of lower leaves and mild chlorotic mottle in middle leaves. The cultivars may possibly develop severe symptoms, which may lead to great economic losses, when coinfected by both SPFMV and SPCSV. As this co-infection is very common worldwide, it is often termed as sweet potato virus disease (SPVD). SPCSV is thus an agriculturally important pathogen of sweet potato, espe- cially because it may break down resistance to unrelated viruses during a co-infection, significantly increasing disease severity. The synergistic effects of SPCSV on other viruses may be due to its interference with RNA silencing because they are often asso- ciated with substantially increased accumulation of coinfecting viruses. Class 1 RNase III encoded by SPCSV has been shown to suppress RNA interference (RNAi)-based antiviral defense system in sweet potato.
The compounds disclosed in this specification may inhibit RNase III encoded by the genomic RNA of SPCSV (CSR3). They may, therefore, be used to treat or prevent SPCSV infections and/or coinfections of SPCSV with one or more other plant pathogenic viruses.
The composition may comprise a compound represented by any one of formulas I to IV, or a salt thereof:
Formula I wherein R1 is H, Cl, F, Br, I, methyl, CF3, COOH, COO-R7, CONHR7, acetyl, alkyl, or aryl;
R2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF3, COO-R7, acetyl, alkyl, or aryl;
R3 is H, Cl, F, Br, I, methyl, ethyl, CF3, COOH, COO-R7, acetyl, SO2NHR7, CONHR7, alkyl, or aryl;
R4 is Br, H, CF3, COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, acetyl, or COO-R7;
R5 is H, acetyl, methyl, ethyl, alkyl, or aryl;
R6 is acetyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, cy- clohexenyl, phenyl, naphthyl, pyridyl, COOH, COO-R7, halogen, meth- oxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, H, or a C6 to C8 aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, -0(C0)R7, methyl, ethyl, propyl, isopropyl, n-bu- tyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclo- hexyl, halogen, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, hydroxyl, nitro, or H; wherein each R7 is independently -CH2CH2CH2COOH, CH2CH2COOH, -CH2COOH, alkyl, aryl, or a Ce to Cs aromatic ring optionally substituted with one or more substituents selected in- dependently from the group consisting of -COOH, -CH2-COOH, -CH2- CH2-COOH, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichlorome- thyl, hydroxyl, nitro, or H; and wherein each alkyl and/or aryl is optionally substituted;
Formula II wherein
R8 to R12 are each independently selected from alkyl, methoxy, ethoxy, acetamido, phenyl, H, and halogen;
R14,and R17 are each independently selected from H, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F,
Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H;
R15 and R16 are each independently selected from H, NH2, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H; and wherein each alkyl is optionally substituted;
Formula III wherein
R18 is H or lower alkyl;
R19,R20,R22,R23,R24, and R25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy, ethoxy, and NR37; H, halogen, methyl, lower alkyl, methoxy, eth- oxy, or
wherein
R26 to R30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy;
R37 is H, methyl, lower alkyl, phenyl, -(CO)CH2CH2CH2CH2CH3, - (CO)CH2CH2CH2CH3, -(CO)CH2CH2CH3, -(CO)CH2CH3, or -(CO)CH3; and wherein each alkyl is optionally substituted;
R32, R33, R34, R35, and R36 are each independently selected from H, OH, methoxy, and ethoxy; and wherein each alkyl is optionally substituted.
The composition may comprise a compound represented by any one of formulas I to IV, or a salt thereof:
Formula I wherein Ri is H, Cl, F, Br, I, methyl, CF3, COOH, COO-R7, alkyl, or aryl;
R2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF3, COO-R7, alkyl, or aryl;
R3 is H, Cl, F, Br, I, methyl, ethyl, CF3, COOH, COO-R7, alkyl, or aryl;
R4 is Br, H, CF3, COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, or COO-R7;
R5 is H, methyl, ethyl, alkyl, or aryl;
R6 is methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, phenyl, COOH, COO-R7, halogen, methoxy, ethoxy, trifluoromethyl, tribro- momethyl, trichloromethyl, H, or a Ce to Ce aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl,
cyclopentyl, cyclohexyl, halogen, methoxy, ethoxy, trifluorome- thyl, tribromomethyl, trichloromethyl, or H; wherein each R7 is independently alkyl or aryl; and wherein each alkyl and/or aryl is optionally substituted;
Formula II wherein
Ri4 and R17 are each independently selected from H, methyl, and lower alkyl;
Ri5 and Ri6 are each independently selected from H, N¾, methyl and lower alkyl; and wherein each alkyl is optionally substituted;
Formula III wherein
R18 is H or lower alkyl;
R19,R20, R22, R23, R24, and R25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy, and ethoxy;
R21 is H, OH, halogen, methyl, lower alkyl, methoxy, eth- oxy, or
wherein
R26 to R30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy; and wherein each alkyl is optionally substituted;
wherein R31 is H, methyl, ethyl, or lower alkyl;
R32, R33, R34, R35, and R36, are each independently selected from H, OH, methoxy, and ethoxy; and wherein each alkyl is optionally substituted.
The composition may be a composition for inhibiting RNase III encoded by SPCSV and/or for reducing the accumulation of at least one plant pathogenic virus in a plant.
The composition may be a composition for treating a plant curatively and/or preventively against symptoms caused by at least one plant pathogenic virus and/or against sweet potato virus dis- ease (SPVD).
The composition may be a crop protection composition.
The composition may further comprise one or more addi- tives, vehicles, formulation auxiliaries, extenders, fillers, and/or surface-active agents. The one or more additives, vehicles, formulation auxiliaries, extenders, fillers, and/or surface-active agents may be agriculturally acceptable. The composition may, al- ternatively or additionally, comprise at least one pesticidally active substance. For example, the at least one pesticidally active substance may comprise or be a pesticidally active substance ef- fective against one or more vectors of plant viruses, such as
aphids and/or whiteflies, or other vector(s) capable of spreading any plant pathogenic virus described in this specification. The pesticidally active substance may be, for example, an insecticide, acaricide, herbicide, fungicide, safener, or growth regulator.
A method for treating a plant curatively and/or preven- tively against at least one plant pathogenic virus and/or against sweet potato virus disease (SPVD) is also disclosed. The method may comprise applying a compound represented by any one of formulas I to IV according to one or more embodiments described in this specification or a salt thereof, or any compound described in this specification or a salt thereof, or a composition according to one or more embodiments described in this specification, to the plant or a part thereof, a cutting for growing the plant, and/or a tuberous root of the plant, or to soil and/or to substrate in which the plant is growing or in which it is desired to grow.
A compound, or a composition comprising a compound, is also disclosed, wherein the compound is capable of inhibiting RNase
III encoded by Sweet potato chlorotic stunt virus (SPCSV) or iden- tified as capable of inhibiting RNase III encoded by SPCSV by a binding assay and/or as capable of reducing the accumulation of at least one plant pathogenic virus in a plant.
The binding assay may comprise or be an assay (or one or more assays) based on fluorescence resonance energy transfer (FRET), microscale thermophoresis (MST) and/or surface plasmon resonance (SPR). Such binding assays are described in detail e.g. in the Examples of the present specification. Further, methods for determining whether a compound or composition is capable of re- ducing the accumulation of at least one plant pathogenic virus in a plant, for example, the accumulation of SPCSV in sweet potato, are also described in detail e.g. in the Examples of the present specification .
The RNase III encoded by Sweet potato chlorotic stunt virus (SPCSV) may have an amino acid sequence set forth in the GenBank accession no. GenBank: ADQ42569.1 and/or in SEQ ID NO: 1.
Use of a compound represented by any one of formulas I to
IV according to one or more embodiments described in this speci- fication, or a salt thereof, or of any compound described in this specification or a salt thereof, or of a composition according to
one or more embodiments described in this specification, for treat- ing a plant curatively and/or preventively against symptoms caused by at least one plant pathogenic virus and/or against sweet potato virus disease (SPVD), is disclosed.
Use of a compound represented by any one of formulas I to IV according to one or more embodiments described in this speci- fication, or a salt thereof, or of any compound described in this specification or a salt thereof, or of a composition according to one or more embodiments described in this specification, for in- hibiting RNase III encoded by SPCSV and/or reducing the accumula- tion of at least one plant pathogenic virus in a plant is also disclosed.
Any embodiments described below are disclosed and may be applicable in the context of the method, the compound, the compo- sition, or the uses according to one or more embodiments described in this specification.
Formula I wherein Ri is H, Cl, F, Br, I, methyl, CF3, COOH, COO-R7, CONHR7, acetyl, alkyl, or aryl;
R2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF3, COO-R7, acetyl, alkyl, or aryl;
R3 is H, Cl, F, Br, I, methyl, ethyl, CF3, COOH, COO-R7, acetyl, SO2NHR7, CONHR7, alkyl, or aryl;
R4 is Br, H, CF3, COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, acetyl, or COO-R7;
R5 is H, acetyl, methyl, ethyl, alkyl, or aryl;
R6 is acetyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, cy- clohexenyl, phenyl, naphthyl, pyridyl, COOH, COO-R7, halogen, meth- oxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, H,
or a C6 to Os aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, -0(C0)R7, methyl, ethyl, propyl, isopropyl, n-bu- tyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclo- hexyl, halogen, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, hydroxyl, nitro, or H; wherein each R7 is independently -CH2CH2CH2COOH, CH2CH2COOH, -CH2COOH, alkyl, aryl, or a C6 to Cs aromatic ring optionally substituted with one or more substituents selected in- dependently from the group consisting of -COOH, -CH2-COOH, -CH2 CH2-COOH, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichlorome- thyl, hydroxyl, nitro, or H; and wherein each alkyl and/or aryl is optionally substituted.
Formula I wherein R7 is H, Cl, F, Br, I, methyl, CF3, COOH, COO-R7, alkyl, or aryl;
R2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF3, COO-R7, alkyl, or aryl;
R3 is H, Cl, F, Br, I, methyl, ethyl, CF3, COOH, COO-R7, alkyl, or aryl;
R4 is Br, H, CF3, COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, or COO-R7;
R5 is H, methyl, ethyl, alkyl, or aryl;
R6 is methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, phenyl, COOH, COO-R7, halogen, methoxy, ethoxy, trifluoromethyl, tribro- momethyl, trichloromethyl, H, or a C6 to Cs aromatic ring optionally
substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclo- pentyl, cyclohexyl, halogen, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, hydroxyl, or H; wherein each R7 is independently alkyl or aryl; and wherein each alkyl and/or aryl is optionally substituted.
R1 may, in some embodiments, be H, Cl, F, Br, or I.
R1 may, in some embodiments, be H or Cl.
R2 may, in some embodiments, be H or COOH.
R3 may, in some embodiments, be H, Cl, F, Br, or I.
R3 may, in some embodiments, be H or Cl.
R4 may, in some embodiments, be Br, H, CF3, COOH, Cl, F, I, or methyl.
R4 may, in some embodiments, be Br, H, CF3, or COOH.
R5 may, in some embodiments, be H or methyl.
R5 may, in some embodiments, be H.
Rg may, in some embodiments, be carboxyphenyl, methylphenyl, fluorophenyl, fluorophenyl, phenyl, ethoxycarbonyl, COOH, isopropyl, substituted aromatic ring, alkyl, or COO-R7.
Rg may, in some embodiments, be carboxyphenyl (0-, m-, or p-carboxyphenyl, in particular, p-carboxyphenyl), methylphenyl (o- , m-, or p-methylphenyl, in particular, p-methylphenyl), fluoro- phenyl (0-, m-, or p-fluorophenyl, in particular, p-fluorophenyl or o-fluorophenyl), phenyl, ethoxycarbonyl, COOH, or isopropyl.
R7 may, in some embodiments, be alkyl, such as methyl or ethyl.
Formula I wherein Riis H, Cl, F, Br, or I;
R2 is H or COOH;
R3 is H, Cl, F, Br, or I;
R4 is Br, H, CF3, COOH, Cl, F, I, or methyl;
R5 is H, or methyl;
R6 is p-carboxyphenyl, p-methylphenyl, p-fluorophenyl, o- fluorophenyl, phenyl, ethoxycarbonyl, COOH, isopropyl, substituted aromatic ring, alkyl, or COO-R7; wherein each R7 is independently alkyl or aryl; and wherein each alkyl and/or aryl is optionally substituted. Examples of compounds represented by formula I include (but are not limited to) the following:
Examples of compounds represented by formula I include (but are not limited to) the following: p-{10-Bromo-8-azatricyclo [7.4.0.02 ' 6]trideca- 1(13),3,9,ll-tetraen-7-yl jbenzoic acid (SMILES notation OC (=0)clccc (ccl)ClNc2c (Br)cccc2C2C=CCC12; also referred to as FIMM022230 in this specification);
7- (p-Tolyl)-8-azatricyclo[7.4.0.02 ' 6]trideca- 1(13),3,9,ll-tetraene-12-carboxylic acid (SMILES notation Cclccc(ccl) [CH]1NC2CCC (cc2[CH]2C=CC [CH]12)C (0)=0; also referred to as FIMM031755 in this specification);
(2R,6S,1R)-7-(p-Tolyl)-8-azatricy- clo[7.4.0.02 ' 6]trideca-l(13),3,9,11-tetraene-12-carboxylic acid
(SMILES notation
Cclccc(ccl) [C@@H]1NC2CCC (cc2[C@@H]2C=CC [C@H]12)C (0)=0; also re- ferred to as FIMM031755 in this specification);
7-Phenyl-8-azatricyclo [7.4.0.02 ' 6]trideca-l(13),3,9,11- tetraene-12-carboxylic acid (SMILES notation
OC (=0)clccc2NC (C3CC=CC3c2cl)clcccccl; also referred to as
FIMM005536 in this specification);
7- (p-Fluorophenyl)-8-azatricyclo[7.4.0.02 ' 6]trideca- 1(13),3,9,ll-tetraene-12-carboxylic acid (SMILES notation
OC (=0)clccc2NC (C3CC=CC3c2cl)clccc (F)ccl; also referred to as FIMM051696 in this specification);
7- (o-Fluorophenyl)-8-azatricyclo[7.4.0.02 ' 6]trideca- 1(13),3,9,ll-tetraene-12-carboxylic acid (SMILES notation
OC (=0)clccc2NC (C3CC=CC3c2cl)clccccclF; also referred to as
FIMM056930 in this specification);
7-Ethoxycarbonyl-8-azatricyclo [7.4.0.02 ' 6]trideca- 1(13),3,9,ll-tetraene-12-carboxylic acid (SMILES notation
CC0C(=0)[C@@H]1NC2CCC (cc2[C@@H]2C=CC [C@H]12)C (0)=0; also referred to as FIMM116926 in this specification);
10- (Trifluoromethyl)-8-azatricyclo[7.4.0.02 ' 6]trideca- 1(13),3,9,ll-tetraene-7-carboxylic acid (SMILES notation
OC (=0)ClNc2c(cccc2C(F)(F)F)C2C=CCC12; also referred to as FIMM005613 in this specification);
10- (Trifluoromethyl)-8-azatricyclo[7.4.0.02 ' 6]trideca- 1 (13),3,9,ll-tetraene-7-carboxylic acid (SMILES notation OC (=0)ClNc2ccc(Br)cc2C2C=CCC12; also referred to as FIMM025159 in this specification);
7-Isopropyl-8-azatricyclo[7.4.0.02 ' 6]trideca- 1 (13),3,9,11-tetraene-lO-carboxylic acid (SMILES notation
CC (C)ClNc2c(cccc2C(O)=0)C2C=CCC12; also referred to as FIMM029702 in this specification); ll,13-Dichloro-8-azatricyclo[7.4.0.02 ' 6]trideca- 1 (13),3,9,ll-tetraene-7-carboxylic acid (SMILES notation
OC (=0)ClNc2cc(Cl)cc(Cl)c2C2C=CCC12; also referred to as FIMM029703 in this specification). In case there are discrepancies between the naming and/or structure of a compound disclosed in this specification and its SMILES notation, the SMILES notation may be considered correct. The SMILES notation has been originally described e.g. in Anderson E, Veith GD, Weininger D (1987). SMILES: A line notation and com- puterized interpreter for chemical structures. Duluth, MN: U.S. EPA, Environmental Research Laboratory-Duluth . Report No. EPA/600/M-87/021.
Examples of compounds represented by formula I include (but are not limited to) the following:
In an embodiment, the compound may be a compound repre- sented by formula II, or a salt thereof:
wherein
R8 to R12 are each independently selected from alkyl, methoxy, ethoxy, acetamido, phenyl, H, and halogen;
R14, and R17 are each independently selected from H, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F,
Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H;
R15 and R16 are each independently selected from H, NH2, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H; and wherein each alkyl is optionally substituted.
Formula II wherein
R14 and R17 are each independently selected from H, methyl, and lower alkyl;
R15 and R16 are each independently selected from H, NH2, methyl and lower alkyl; and wherein each alkyl is optionally substituted.
R8 to R12 may, in some embodiments, be each independently selected from alkyl, methoxy, H, and halogen.
R8 and Rio may, in some embodiments, both be halogen.
R8 and R11 may, in some embodiments, both be lower alkyl. R8 and R11 may, in some embodiments, both be methyl or ethyl.
R8 and R11 may, in some embodiments, both be methyl.
R9 and R11 may, in some embodiments, both be halogen.
R10 and R11 may, in some embodiments, both be methoxy or ethoxy, or they may be independently selected from H, methoxy and ethoxy.
R15 may, in some embodiments, be H.
R16 may, in some embodiments, be H or NH2.
Examples of compounds represented by formula II include (but are not limited to) the following:
2- (2H-1,2,4-Triazol-3-ylthio)-1-(2,5-xylyl)-1-ethanone (SMILES notation Cclccc(C)c(cl)C (=0)CSclncn[nH]1; also referred to as FIMM027745 in this specification);
1- (3,4-Dimethoxyphenyl)-2-(2H-1,2,4-triazol-3-ylthio)- 1-ethanone (SMILES notation COclccc(cclOC)C (=0)CSclnnc[nH]1; also referred to as FIMM027749 in this specification);
2- (5-Amino-4H-l,2,4-triazol-3-ylthio)-1-(2,4-dichloro- phenyl)-1-ethanone (SMILES notation
Nclnnc (SCC (=0)c2ccc(Cl)cc2Cl)[nH]l; also referred to as FIMM017990 in this specification).
Formula III wherein
R18 is H or lower alkyl;
R19, R2 0 , R22 , R23 , R24 , and R25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy, ethoxy, and
NR37;
R26 to R30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy;
R37 is H, methyl, lower alkyl, phenyl, -(CO)CH2CH2CH2CH2CH3, - (CO)CH2CH2CH2CH3, -(CO)CH2CH2CH3, -(CO)CH2CH3, or -(CO)CH3; and wherein each alkyl is optionally substituted.
Formula III wherein
R18 is H or lower alkyl;
R19, R2 0 , R22 , R23 , R24 , and R25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy and ethoxy;
R26 to R30 are each independently selected from H , OH, halogen, lower alkyl, methoxy and ethoxy; and wherein each alkyl is optionally substituted.
R18 may, in some embodiments, be H.
R19, R2 0 , R22 , R23 , R24 , and R25 may, in some embodiments, be each independently selected from H and OH.
R21 may, in some embodiments, be H.
R26 to R30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy.
R26 to R30 are each independently selected from H and OH. Examples of compounds represented by formula III include (but are not limited to) the following:
Examples of compounds represented by formula III include (but are not limited to) the following:
6-Hydroxy-l-naphthalenesulfonic acid (SMILES notation Oclccc2c (cccc2cl)S([0-])(=0)=0; also referred to as FIMM072577 in this specification);
4- [(E)-2,4-Dihydroxyphenylazo]-3-hydroxy-l-naphtha- lenesulfonic acid (SMILES notation
0=S(=0) (0)c2cc(0)c(/N=N/clccc(0)cclO)c3ccccc23; also referred to as FIMM116487 in this specification).
In an embodiment, the compound may be a compound repre- sented by formula IV, or a salt thereof:
wherein R31 is H, methyl, ethyl, or lower alkyl; and R32 , R33 , R34 , R35 , and R3 6 are each independently selected from H, OH, methoxy, and ethoxy.
R31 may, in some embodiments, be H or methyl.
R32 , R33 , R34 , R35 , and R3 6 may, in some embodiments, be each independently selected from H, OH, and methoxy. Examples of compounds represented by formula IV include
Examples of compounds represented by formula IV include (but are not limited to) the following:
10-Methyl-10-azatetracyclo [7.7.1.02 ' 7.O13 ' 17]heptadeca- 1(16),2,4,6,13 (17),14-hexaene-3,4-diol (SMILES notation
CN1CCC2=C3C1CC4=CC=C (O)C(=0403=00=02)O; also referred to as FIMM000096 or apomorphine in this specification); (R)-10-Methyl-10-azatetracyclo[7.7.1.O2 ' 7.O13 ' 17]hepta- deca-1(16),2,4,6,13 (17),14-hexaene-3,4-diol (SMILES notation
C1C (=C2)C (C (=CC=C3)C (=C3C3)[C@@H]IN (C)C3)=C (C (0)=C2)0; also re- ferred to as FIMM000096 or apomorphine in this specification);
15,16-Dimethoxy-10-azatetracy clo[7.7.1.02 ' 7.013 ' 17]hepta- deca-1 (16),2,4,6,13(17),14-hexaen-4-ol (SMILES notation
C0C1=CC2=C3C (CC4=C (C=C (O)C=C4)C3=C10C)NCC2; also referred to as FIMM011744 in this specification);
4.16-Dimethoxy-l0-methyl- 1O-azatetracy- clo[7.7.1.O2 ' 7 .O13 ' 17]heptadeca-1(16),2,4,6,13(17),14-hexaene-5,15- diol (SMILES notation
Cl (C (C2)N3C)C (C (C2=CC20)=CC=20C)=C (OC)C (=CC=1CC3)O);
4.15.16-Trimethoxy-10-methyl-10-azatetracy- clo[7.7.1.O2 ' 7 .O13 ' 17]heptadeca-1(16),2,4,6,13(17),14-hexaen-3-ol) (SMILES notation
Cl (N (C2)C)CC (=C3)C (C (C1=C1C2)=C (OC)C (=C1)OC)=C (C (=C3)OC)O).
In the context of this specification (and in the context of any one of the formulas I to IV), the term "halogen" may be understood as referring to F, Cl, Br, and/or I.
In the context of this specification (and in the context of any one of the formulas I to IV), the term "lower alkyl" may be understood as referring to an unbranched (i.e. straight chained) or branched C1-C4 alkyl, such as an unbranched or branched C1, C2, C3 or C4 alkyl. Examples of lower alkyls include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, and tert-butyl.
In the context of this specification (and in the context of any one of the formulas I to IV), the term "alkyl" may be understood as referring to a monovalent moiety obtained or obtain- able by removing a hydrogen atom from a carbon atom of a hydro- carbon compound, which may be aliphatic or alicyclic, and which may be saturated. Thus, the term "alkyl" includes the sub-class cycloalkyl, and the like. In an embodiment, the term "C1-12 alkyl" means an alkyl moiety having from 1 to 12 carbon atoms.
In the context of this specification (and in the context of any one of the formulas I to IV), the term "aryl" may be understood as referring to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. For example, each ring may have from 5 to 8 ring atoms.
In this context, the prefixes (e.g. C3-20, C5-8, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C5-6 aryl" as used herein means an aryl group having 5 or 6 ring atoms.
The ring atoms may be all carbon atoms, as in "carboaryl groups". Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (Cis), and pyrene (C16)· In an embodiment, the aryl is phenyl.
Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-lH- indene) (C9), indene (C9), isoindene (C9), tetraline (1,2,3,4- tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and aceanthrene (C16)·
Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroaryl groups". Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:
N1: pyrrole (azole) (C5), pyridine (azine) (C6);
O1 : furan (oxole) (C5);
S1: thiophene (thiole) (C5);
N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6);
N2O1 : oxadiazole (furazan) (C5);
N3O1 : oxatriazole (C5);
N1S1: thiazole (C5), isothiazole (C5);
N2 : imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6);
N3 : triazole (C5), triazine (C6); and,
N4 : tetrazole (C5).
Examples of heteroaryls which comprise fused rings, include, but are not limited to:
C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1),
benzisoxazole (N1O1), benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S);
C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), benzodioxan (O2) quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4);
C11 (with 2 fused rings) derived from benzodiazepine (N2);
C13 (with 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); and,
C14 (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O2), phenoxathiin
(O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N2S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).
In the context of this specification (and in the context of any one of the formulas I to IV), the terms "substituted alkyl" and/or "substituted aryl" may be understood as referring to an alkyl or aryl substituted by one or more substituents selected from -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, halogen, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, or H. Each R7 may be independently alkyl or aryl, wherein each alkyl and/or aryl may be optionally substi- tuted.
The compound may be selected from the following, and/or their salts. In other words, the composition may, alternatively or additionally, comprise a compound selected from the following, and/or their salts:
CCCClNc2ccc (cc2C2C=CCC12)C (=0)0; also referred to as FIMM004463 in this specification);
(SMILES nota- tionOC (=0)clcccc2clNC (C1CC=CC12)clcccccl; also referred to as FIMM005356 in this specification); ((SMILES notation
OC (=0)CNC (=0)clccc2c(cl)C1C=CCC1C (N2)clccc(ccl)F; also referred to as FIMM005542 in this specification);
(SMILES notation
OC (=0)clccc2c(cl)C1C=CCC1C (N2)C1CCC=CC1; also referred to as
OC (=0)clccc2c(cl)C1C=CCC1C (N2)clcccc(cl)Br; also referred to as FIMM006696 in this specification);
(SMILES notation
CC (=0)clccc2c(cl)C1C=CCC1C (N2)clcccccl[N+](=0)[0-]; also referred to as FIMM006830 in this specification);
(SMILES notation
OC (=0)clcccc2clNC (C1CC=CC12)clccccclF; also referred to as FIMM007231 in this specification);
(SMILES notation
OC (=0)clccc(ccl)ClNc2c(cc(cc2C2C=CCC12)F)F; also referred to as FIMM021251 in this specification);
(SMILES notation
OC (=0)clccc2c(cl)C1C=CCC1C (N2)clcccc2cccccl2; also referred to FIMM025241 as in this specification);
CC (C)ClNc2ccc(cc2C2C=CCC12)C (=0)0; also referred to FIMM025818 as in this specification);
(SMILES notation
Cclc2c(ccclC (=0)0)[C@@H]1C=CC[C@@H]1[C@@H](N2)clccc(ccl)Cl; also referred to as FIMM029749 in this specification);
, (SMILES notation CC (C)clccc(ccl)[C@@H]1NC2CCC(cc2[C@@H]2C=CC[C@H]12)C (=0)O; also referred to as FIMM030753 in this specification); (SMILES notation
OC (=0)clccc2c(cl)[C@@H]1C=CC[C@@H]1[C@@H](N2)clcccc(cl)Cl; also referred to as FIMM030754 in this specification);
(SMILES notation
OC (=0)clccc(ccl)[C@@H]!Nc2ccccc2[C@@H]2C=CC[C@H]12; also referred to as FIMM030756 in this specification);
, (SMILES notation
Cclcc(c2c (cl)[C@@H]1C=CC[C@@H]1[C@@H](N2)clccc(ccl)C (=0)0)C; also referred to as FIMM030757 in this specification);
, (SMILES notation
Cclccc2c (clC)N [C@H]([C@H]1CC=C[C@H]12)clccc(ccl)C (=0)0; also re- ferred to as FIMM030758 in this specification);
(SMILES notation
CCclcccc2clN [C@H]([C@H]1CC=C[C@H]12)clccc(ccl)C (=0)0; also re- ferred to as FIMM030759 in this specification);
, (SMILES notation
OC (=0)clccc(ccl)[C@@H]1NC2C (cccc2[C@@H]2C=CC[C@H]12)F; also re- ferred to as FIMM030760 in this specification); (SMILES notation
OC (=0)clccc2c(cl)[C@@H]1C=CC[C@@H]1[C@@H](N2)clccccclBr; also re- ferred to as FIMM030764 in this specification);
(SMILES notation
Cclc2c(ccclC (=0)0)[C@@H]1C=CC[C@@H]1[C@@H](N2)clcccc(cl)Cl; also referred to as FIMM030766 in this specification);
(SMILES notation
Fclcccc2clN [C@H]([C@H]1CC=C[C@H]12)clccccnl; also referred to as FIMM031757 in this specification);
, (SMILES notation
Cclccc(c2clN [C@H]([C@H]1CC=C[C@H]12)clccc(ccl)Cl)C (=0)0; also re- ferred to as FIMM031758 in this specification);
(SMILES notation OC (=0)clcccc2clNC (C1CC=CC12)clccc(ccl)[N+](=0)[0-]; also referred to as FIMM051697 in this specification);
, (SMILES notation
CC(=0)Oclccc (c(cl)OC(=0)C)ClNc2ccc(cc2C2C=CCC12)C(=0)O; also re- ferred to as FIMM055742 in this specification);
(SMILES notation CC(=0)Oclccc (c(cl)OC(=0)C)ClNc2c(cccc2C(=0)O)C2C=CCC12; also re- ferred to as FIMM056347 in this specification);
(SMILES notation OC(=0)clccc2c (cl)C1C=CCC1C(N2)clccccclF; also referred to as FIMM056930 in this specification);
, (SMILES notation
OC(=0)clcccc2clNC (C1CC=CC12)clcccc(cl)Br; also referred to as FIMM059187 in this specification);
, (SMILES notation
OC(=0)clcccc2clNC (C1CC=CC12)clccc(ccl)F; also referred to as FIMM102447 in this specification);
(SMILES notation
COclcccc (cl)NS(=0)(=0)clccc2c(cl)C1C=CCC1C(N2)clccc(ccl)C(=0)0; also referred to as FIMM116654 in this specification);
, (SMILES notation
OC (=0)clccc2c(cl)[C@@H]1C=CC[C@@H]1[C@@H](N2)clccccnl; also re- ferred to as FIMM117470 in this specification);
(SMILES notation
Cclccc (cclC)C (=0)CSclnnc[nH]1 also referred to as FIMM007089 in this specification);
(SMILES notation
Cclccc (ccl)NlC (=0)CC (Sc2nnc[nH]2)C1=0; also referred to as
CCclnnc ([nH]1)SCC (=0)clccc(ccl)Cl; also referred to as FIMM011010 in this specification);
(SMILES notation
CC (C)Cclnnc([nH]1)SCC (=0)clccc(ccl)Br; also referred to as
Brclccc(ccl)C(=0)CSclnc(n[nH]l)-clcccccl; also referred to as
Brclccc (ccl)C (=0)CSclnnc[nH]1; also referred to as FIMM027143 in this specification);
(SMILES notation
Cclccc (c(cl)C)C (=0)CSclnnc[nH]1; also referred to as FIMM027744 in this specification);
, (SMILES notation
Clclccc (ccl)C (=0)CSclnnc[nH]1; also referred to as FIMM027746 in this specification);
, (SMILES notation
Clclccc (cclCl)C (=0)CSclnnc[nH]1; also referred to as FIMM027747 in this specification);
, (SMILES notation 0=C (CSclnnc[nH]1)clcccsl; also referred to as FIMM027748 in this specification);
, (SMILES notation Cclnnc([nH]1)SCC (=0)clccc(ccl)Cl; also referred to as FIMM028249 in this specification);
, (SMILES notation
Cclnnc([nH]1)SCC (=0)clccc(ccl)F; also referred to as FIMM028250 in this specification);
(SMILES notation
Cclcc(c (c(cl)C)C (=0)CSclnnc[nH]1)C; also referred to as FIMM028260 in this specification);
, (SMILES notation
CC (C)clccc(ccl)C (=0)CSclnnc[nH]1; also referred to as FIMM028263 in this specification);
(SMILES notation Ccl[nH]c2ccccc2clC (=0)CSclnnc[nH]1; also referred to as FIMM032184 in this specification);
, (SMILES notation 0=C (CSclnnc[nH]1)clcccccl; also referred to as FIMM034263 in this specification);
(SMILES notation
Nclnc (n[nH]1)SCC (=0)clccc(ccl)Cl; also referred to as FIMM047835 in this specification);
, (SMILES notation
CC (=0)Nclccc(ccl)C (=0)CSclnnc[nH]1; also referred to as FIMM058866 in this specification);
(SMILES notation
Clclccc (ccl)C (=0)CSclcn[nH]nl; also referred to as FIMM081375 in this specification);
(SMILES notation
Cclccc (ccl)C (=0)CSclnc(n[nH]1)-clccccclCl; also referred to as FIMM095850 in this specification);
, (SMILES notation
0=C (CSclnnc([nH]1)-clcccccl)clccc(ccl)-clcccccl; also referred to as FIMM095857 in this specification);
, (SMILES notation
COclccc (ccl)C(=0)CSclnnc(nl-clcccccl)-clccc(c(cl)0C)0C; also re- ferred to as FIMM105675 in this specification);
(SMILES notation
OS(=0)(=0)clcccc2cccc (cl2)Nclcccccl; also referred to as FIMM003918 in this specification);
r (SMILES notation
Oclccc (ccl)ClNc2cccc3cccc (c23)Nl; also referred to as FIMMO32716 in this specification); r (SMILES notation
CCCCC (=0)Nclcccc2cccc (cl2)S(=0)(=0)0; also referred to as FIMM094180 in this specification);
r (SMILES notation
0clcc(cc2cc(cccl2)Nclcccccl)S(=0) (=0)0; also referred to as FIMM116826 in this specification);
(SMILES notation
0clcc(cc2ccc(ccl2)Nclcccccl)S(=0) (=0)0; also referred to as
FIMM116854 in this specification);
(SMILES notation CC (=0)Nclcccc2cc (cccl2)O; also referred to as FIMM116916 in this specification).
Examples of compounds represented by formula III include (but are not limited to) the following:
The at least one plant pathogenic virus may comprise or be at least one of Sweet potato chlorotic stunt virus (SPCSV) or Sweet potato feathery mottle virus (SPFMV).
The at least one plant pathogenic virus may comprise or be SPCSV, optionally synergistic with at least one other plant pathogenic virus.
In the context of this specification, the term "syner- gistic with at least one other plant pathogenic virus" may be understood as referring to SPCSV co-infecting the plant with the at least one other plant pathogenic virus. When SPCSV is syner- gistic with the at least one other plant pathogenic virus, i.e. when SPCSV co-infects the plant with the at least one other plant pathogenic virus, the viruses may, at least in some embodiments, synergistically cause symptoms in the plant that may be more severe than symptoms caused by either (or single) plant pathogenic virus alone.
SPCSV may be synergistic with one or more Potyviruses (SPFMV, SPLV and/or SPMSV), Cucumoviruses (CMV), Ipomoviruses (SPMMV), Carlaviruses (SPCFV and/or C-6 virus), and/or Cave- moviruses (SPVCV and/or SPCV).
Examples of Potyviruses with which SPCSV may be syner- gistic include SPFMV, SPLV, i.e. Sweet potato latent virus, and SPMSV, i.e. Sweet potato mild speckling virus.
Examples of Cucumoviruses with which SPCSV may be syner- gistic include CMV, i.e. Cucumber mosaic virus.
Examples of Ipomoviruses with which SPCSV may be syner- gistic include SPMMV, i.e. Sweet potato mild mottle virus.
Examples of Carlaviruses with which SPCSV may be syner- gistic include SPCFV, i.e. Sweet potato chlorotic fleck virus, and C-6 virus, i.e. sweet potato C6 virus.
Examples of Cavemoviruses with which SPCSV may be syner- gistic include SPVCV, i.e. Sweet potato vein clearing virus, and SPCV, i.e. Sweet potato caulimo-like virus.
SPCSV may be synergistic with SPFMV and optionally with one or more of other Potyviruses (SPLV and/or SPMSV), Cucumoviruses (CMV), Ipomoviruses (SPMMV), Carlaviruses (SPCFV and/or C-6 vi- rus), and/or Cavemoviruses (SPVCV and/or SPCV).
The plant may be a plant of Ipomoea spp.
The plant may be sweet potato (Ipomoea batatas L.). Other plants of Ipomoea spp. may include e.g. species used as crops, such as water spinach (I. aquatica), whitestar potato (I. lacu- nosa), Australian bush potato (I. costata); and/or species that are used as ornamental plants, such as heavenly blue morning glory (I. violacea), tropical white morning glory (I. alba), common morning glory (I. purpurea) and other morning glories. Other Ipomoea species may also be contemplated.
The compound represented by any one of formulas I to IV according to one or more embodiments described in this specifica- tion or a salt thereof, or the composition according to one or more embodiments described in this specification, may be applied to the plant or a part thereof, a cutting for growing the plant, and/or a tuberous root of the plant, or to soil and/or to substrate in which the plant is growing or in which it is desired to grow. They may be applied e.g. using various application types described below, such as foliar spray, and/or by dipping or coating a cutting (such as a stem-cutting or vine-cutting). They may, additionally or alternatively, be applied in combination with cryotherapy of shoot tips, e.g. before or after the cryotherapy. It may be con- sidered as a complementary approach to reduce viral load, e.g. before cryopreservation.
The term "cryotherapy" may be understood as referring to a treatment in which a part of the plant, such as a shoot tip, is exposed briefly to liquid nitrogen, so as to reduce the number of plant pathogens, such as plant pathogenic viruses, present in the part of the plant. An example of such cryotherapy is described in Wang et al., Annals of Applied Biology 2009, 154(3), 351-363.
The compound or the composition may be applied e.g. by band application, by basal application, by broadcast application, by directed spray, by foliar spray, by rope-wick or wiper appli- cation, by stem application, by drench/drip application (chemiga- tion), or by injection to the plant.
The compound or the composition may be applied e.g. by soil application, by soil incorporation, by soil injection, by space treatment, or by spot treatment to soil and/or to substrate in which the plant is growing or in which it is desired to grow.
The substrate in which the plant is growing or in which it is desired to grow may comprise or be any suitable solid or liquid substrate. Examples of substrates may include pumice, py- roclastic material, synthetic organic substrate, organic sub- strate, and liquid substrate.
In the context of this specification, the term "treating a plant curatively against at least one plant pathogenic virus and/or against sweet potato virus disease (SPVD)" may be understood as referring to treating the plant that is infected by the at least one plant pathogenic virus and/or exhibits SPVD. For example, the plant may be treated to alleviate the symptoms caused by the at least one plant pathogenic virus and/or by SPVD.
In the context of this specification, the term "treating a plant preventively against at least one plant pathogenic virus and/or against sweet potato virus disease (SPVD)" may be understood as referring to treating the plant to prevent infection by the at least one plant pathogenic virus and/or to prevent SPVD.
In the context of this specification, the term "sweet potato virus disease (SPVD)" may be understood as referring to a plant disease caused by co-infection by both SPFMV and SPCSV, for example in a plant of Ipomoea spp., such as in sweet potato.
EXAMPLES
Reference will now be made in detail to various embodiments, an example of which is illustrated in the accompanying drawings.
The description below discloses some embodiments in such detail that a person skilled in the art is able to utilize the embodiments based on the disclosure. Not all steps or features of the embodiments are discussed in detail, as many of the steps or features will be obvious for the person skilled in the art based on this specification.
EXAMPLE 1
CSR3 characteristics for FRET-Based High-Throughput Screening (HTS) for viral RNase III Inhibitors
For the development of an HTS assay, His-tagged CSR3 (SEQ ID NO: 2) and its double-mutant CSR3-A (D37A, D44A; SEQ ID NO: 3) were expressed in E. coli and purified with Nickel-nitrilotri- acetic acid (Ni-NTA) agarose.
Protein expression and purification
C-terminal His-tagged CSR3 (GenBank: ADQ42569.1; SEQ ID NO: 2) and CSR3-A (D37A, D44A; SEQ ID NO: 3) were expressed in E. coli from the plasmid pETlld (Kreuze et al., 2005: J Virol, 79, 7227-7238). Bacterial cultures were grown under selection with ampicillin (100 yg/ml) and chloramphenicol (25 yg/ml) at 37°C for 2 h. Recombinant proteins were induced by adding 0.1 mM (final concentration) of isopropyl b-d-l-thiogalactopyranoside (IPTG) into the culture medium and growing for 4 h. Bacterial cells were lysed in lysis buffer (10 mM imidazole) supplemented with 1 tablet of protease inhibitor cocktail (complete ULTAR Tablets, mini, Roche, Basel, Switzerland) per 10 ml lysis buffer and 1 mg/ml lysozyme (Sigma-Aldrich, St. Louis, MO, USA) while incubating for 2 h on ice. Sonication (50% duty cycle, 4 c 15 s; Branson Sonifier [B150R Cell Disruptor B15]) was used to additionally disrupt cells and degrade nucleic acids.
Ni-NTA agarose gravity-flow chromatography (Qiagen, Venlo, Netherlands) was used to purify His-tagged proteins. The bacterial extract was loaded onto polypropylene columns (Qiagen) and washed successively with wash buffer 1 (50 mM imidazole) and wash buffer 2 (70 mM imidazole). Bound proteins were eluted with elution buffer (500 mM imidazole). Lysis buffer, wash buffer, and elution buffer were made using the His Buffer kit from GE Healthcare (Chicago, IL, USA). Fractions containing high concen- trations of pure protein were collected, subjected to buffer ex- change with a PD MidiTrap G-25 (GE Healthcare), and finally diluted in storage buffer (20 mM MgCl2, 40 mM Tris-HCl, 100 mM NaCl, pH 8.0, 5% glycerol). The Bradford colorimetric method (Protein As- say, Dye Reagent concentrate, Bio-Rad, Hercules, CA, USA) and a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA; 280 nm) were used for protein quantification. Coomassie Bril- liant Blue reagent [10% (v/v) glacial acetic acid, 40% (v/v) meth- anol, 1% (w/v) Coomassie Brilliant Blue G] was used to visualize proteins in gels following SDS-PAGE.
Western blotting
Proteins were resolved via Tris-glycine SDS-PAGE (12% polyacrylamide) and transferred to polyvinylidene difluoride mem- brane (GE Healthcare) by electroblotting. After SDS-PAGE electrob- lotting, gels were stained with Coomassie Brilliant Blue to confirm that the majority of proteins were transferred. Each membrane was then incubated for 60 min in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KC1, 10 mM Na2HP04, 2 mM KH2P04, pH 8) containing 0.1% (w/v) Tween-20 (PBS-Tween) with 5% non-fat dried milk and washed 3 x 10 min in PBS-Tween at room temperature. Then, each membrane was incubated for 1 h with a CSR3-specific rabbit poly- clonal antibody (Kreuze et al., 2005: J Virol, 79, 7227-7238) diluted 1:1000 in PBS-Tween containing 2.5% dried milk. After three washes with PBS-Tween, each membrane was soaked for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma-Aldrich) diluted 1:5000, and bands were visualized with the West Pico chem- iluminescence development substrate (Thermo Fisher Scientific). Chemiluminescence was detected with X-ray film (Roche) using an enhanced chemiluminescence kit from GE Healthcare.
The recombinant CSR3 and its mutant were analyzed with SDS-PAGE, revealing a predominant band at ~26 kDa (Fig. 1A). A second round of elution (Fig. 1A) yielded the majority of CSR3, and aliquots of this fraction were made. In addition, western blotting showed that both proteins can exist as mixed monomer, dimer, and tetramer in storage buffer (Fig. IB).
Size-exclusion chromatography coupled with multi-angle light scattering
The oligomerization of CSR3 was also characterized by size-exclusion chromatography with detection using multi-angle light scattering.
Size-exclusion chromatography coupled with multi-angle light scattering was used for characterizing oligomeric states of CSR3. Samples of purified recombinant CSR3 diluted in PBS were loaded onto a Superdex S-20010/300 GL (GE Healthcare) column at 0.5 ml/min with an HPLC system (Shimadzu, Kyoto, Japan) coupled with MiniDAWN TREOS multi-angle light-scattering detector, and Optilab rEX refractive index detector (Wyatt Technology, Santa Barbara, CA, USA). The injection volume was 100 μl per sample, and
chromatography was carried out at 4°C. The concentration of protein in the effluent was measured with a light-scattering detector (493- TS; RI instrument 686-REX, 658 nm; UV instrument SPD-M20A). Data were analyzed with ASTRA 6 software (Wyatt Technology).
The only detectable protein peak at molecular mass 68.93 kDa was larger than that of the theoretical dimer of molecular mass 52 kDa, which could be explained either because most of the CSR3 preparation comprised a mixture of dimers and tetramers in the PBS running buffer, or by the non-spherical nature of the dimer which could cause a disruption during size-exclusion elution (Fig. 1C).
CSR3 activity assay as assessed with agarose gel elec- trophoresis
The CSR3 activity was evaluated using a 200-bp double- stranded RNA (dsRNA) substrate.
The dsRNA molecules (200 bp) were generated using the TranscriptAid T7 High-Yield Transcription kit (Thermo Fisher Sci- entific). To precipitate dsRNA, samples were incubated with 2.5 M ammonium acetate on ice for 15 min and centrifuged at 10,000 x g for 15 min at 4°C. After removing the supernatant, the pellet was washed twice with 70% ethanol, air-dried at room temperature for 10 min, and resuspended in RNase-free water (50 μl). To reanneal RNA as a double strand, samples were incubated at 95°C for 10 min, 65°C for 1 min, and room temperature before storage at -20°C. RNase activity was tested in 20 μl reactions (20 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2,pH 8) containing 300 ng of 200-bp dsRNA and 200 ng enzyme. Each reaction was incubated for 40 min at 37°C before loading on a 1% agarose gel.
This substrate was cleaved to smaller dsRNA fragments in the presence of CSR3 (Fig. ID) but remained intact in the presence of CSR3-A, as was also the case for the control reaction lacking any endoribonuclease (Ctl). These results validated the activity of the purified CSR3 enzyme.
EXAMPLE 2
Labeled small interfering RNA (siRNA) constructs
A 22 bp 2-nt 3'-overhang siRNA labeled with a FAM reporter and BHQ1 quencher (forward: 5' FAM-CGUAGUGGAAGUGGGAGAGGTC-BHQ13' [unlabeled sequence set forth in SEQ ID NO: 4]; reverse: 5' CCU- CUCCCACUUCCACUACGTG 3' [SEQ ID NO: 5]) were synthesized by Metabion (Munich, Germany). Their identity and purity were verified by HPLC. The RNA oligonucleotides were dissolved in annealing buffer (6 mM HEPES pH 7.5, containing 60 mM KC1 and 0.2 mM MgCl2) at a concen- tration of 100 mM, aliquoted in volumes ranging from 10 to 200 μl and stored at -20°C. Before use, siRNA was diluted to 15 mM (200 ng/μl) with annealing buffer, incubated 2 min at 93°C and cooled to room temperature (³30 min).
FRET assay development
The HTS assay was designed to maximize siRNA cleavage by CSR3. A donor-quencher fluorogenic siRNA was used as the substrate for CSR3. To optimize the ratio of enzyme to substrate, a CSR3 titration assay was carried out by mixing six different concen- trations of CSR3 (0-1150 nM) with 375 nM labeled siRNA according to results obtained from a preliminary test. To test the repro- ducibility and precision of the assay, full plates containing only negative (50 nM CSR3) and positive reactions (lacking CSR3) were tested with 375 nM labeled siRNA. Enzyme and substrate were pre- pared separately at 2x final concentration, and then 10 μl of each was dispensed into every well with an automated dispenser (MultiFlo FX with single-channel RAD-cassettes; BioTek, Winooski, VT, USA). Plates were 384-well black flat-bottom microplates (#3544, Corn- ing, NY, USA).
FRET assay setup
The HTS assay was based on FRET, in which CSR3 cleaves a labeled siRNA and generates a fluorescent signal (Fig. 2A, B).
The 2-nt overhang of a 22-bp siRNA labeled with a fluor- ophore reporter (FAM) and a quencher (HBQ1) at the 5' and 3' end, respectively, of the sense-strand was used as substrate. Fluores- cence was acquired with excitation/emission of 485/520 nm, re- flecting the unquenched fluorescence intensity of the reporter. Two reaction conditions were used for the negative and positive controls to normalize CSR3-inhibition data acquired for all com- pounds. For the negative control, cleavage of the labeled siRNA by CSR3 disrupts the energy transfer from the donor to the receptor
and hence loss of FRET quenching, allowing the detection of the fluorescence emission of the reporter (Fig. 2A). For the positive control (or blank control), the reaction was carried out either with the catalytically inactive CSR3-A or in the absence of any added enzyme, so that labeled siRNA integrity was maintained and fluorescence emissions from the reporter remained quenched by HBQ1 (Fig. 2B). There was a clear increase in fluorescence in the ab- sence of FRET (i.e., negative controls), whereas fluorescence re- mained stable over time in the presence of FRET (i.e., positive controls) (Fig. 2C). As both positive controls showed similar trends, in order to keep the HTS process simple no enzyme condition was applied as the positive control in the following optimization and screening assays. Moreover, these two control conditions were validated by analysis of the reaction products with 2% agarose gel electrophoresis (Fig. 2D). These results indicated that the design of the assay was suitable for HTS.
Data analysis
The endoribonuclease activity of CSR3 was calculated with slope, representing fluorescence changes in function of assay time (s), using MARS Data Analysis software (BMG Labtech). In addition, the Z' of the assay was calculated according to Eq. A (Zhang et al., 1999, J Biomol Screen, 4, 67-73), which was used to evaluate the suitability of the method during assay development, optimiza- tion, and screening. Moreover, the signal-to-noise ratio and sig- nal-to-background ratio were also used to evaluate the quality of each assay: signal-to-noise ratio = (μc_ - μc+) / oc+, and the signal-to-background ratio = μc_ / μc+ .
Mean of the positive control (μc+) and negative control (μc-). Standard deviation of positive control (oc+) and negative control (oc-).
The percentage of inhibition (PI) of each compound for CSR3 was calculated according to Eq. B. A PI threshold of 30% was used as the cutoff value for one concentration HTS. Furthermore, the dose-response curves of PI in function of compounds' concentra- tion were evaluated with DSS according to Yadav et al. (2014),
Mean of the slope of the positive control (Sc+), Mean of the slope of the negative control (Sc-), and Mean of the slope of samples (Ss).
The kinetic constant (Kd) for CSR3 was calculated using the three-parameter Michaelis-Menten model (MM.3) included in the R package dcr (Ritz et al., 2015, PLoS One, 10, e0146021). The statistical significance of differences between values was as- sessed with one-way ANOVA using the aov function in the R package.
CSR3 titration assay
The rate of increase in fluorescence was dependent on the amount of enzyme and labeled siRNA in the reaction. Fluorescence measurements (Fig. 3A) showed that, compared with a low concen- tration, a high concentration of CSR3 led to a rapid increase and higher fluorescence at the beginning of the kinetic measurement. To quantify the increase in fluorescence, the slopes (fluorescence changes over second time which is depicting the reaction rate of CSR3) were calculated between all neighboring detection cycles. The slopes were used to select the optimal detection time for each CSR3 concentration tested. The results suggested that the maximal initial slope for the concentrations 144 nM, 72 nM, and 36 nM of CSR3 occurred after 5, 10, and 14 reaction cycles, respectively (Fig. 3B), indicating that a lower concentration of enzyme resulted in a longer period during which kinetics could be monitored. A very high kinetic rate was observed with 288 nM and 575 nM CSR3 concentrations during the first cycle (shown for 288 nM in Fig. 3B), making it impossible to monitor the reaction at high enzyme concentrations, i.e., the labeled siRNA was cleaved before fluo- rescence could be monitored. Furthermore, maximal slopes were lin- early correlated with CSR3 concentration (Fig. 3C), indicating that the initial velocity of the reaction correlated positively with CSR3 concentration with a fixed concentration of substrate. Slopes of the positive and negative controls were used to evaluate the suitability of the HTS with the parameter Z prime (Z', see equation above in Data analysis) and its threshold is usually 0.5. The Z1 values with five concentrations and three replicates (Table 1) suggested that an enzyme concentration ranging from 72 to 144 nM could be used because the resulting Z' values were >0.9.
Therefore, the final concentration of enzyme 100 nM CSR3 and sub- strate 375 nM siRNA were used in the HTS assay. Moreover, these two control conditions indicated that both the labeled siRNA and enzyme were stable at room temperature (25°C), an important aspect for HTS given that 20 assay plates were required for the screening and each of them had to be run with 12 detection cycles.
Table 1 Calculated Z' values for the five different CSR3 concentrations used in the three replicates over 12 cycles.
EXAMPLE 3
Primary HTS screen and a dose-response screen of CSR3 HTS assay setup
For the HTS assay, plates (10 mM compound in each well) were prepared with an Echo® liquid handler (Labcyte, San Jose, CA, USA) at the FIMM (Institute for Molecular Medicine Finland) High- Throughput Biomedicine unit. CSR3 and annealed siRNA were diluted to 200 nM and 750 nM, respectively, in reaction buffer (50 mM Tris- HC1, 125 mM NaCl, 25 mM MgCl2, pH 8.0). CSR3 solutions were dis- pensed into the wells of plates (10 μl CSR3 per well). Plates were incubated at room temperature for 15 min with shaking (450 rpm). Subsequently, 10 μl substrate was dispensed per well. The final CSR3 and substrate concentrations were 100 nM and 375 nM, respec- tively. The dose-response assay with six compound concentrations (range, 1.25 nM to 50 mM) was carried out with the same conditions. Plates were sealed, centrifuged briefly, and then immediately an- alyzed with a PHERAstar FS (BMG Labtech, Ortenberg, Germany) with
a fluorescence-intensity optic module (excitation at 485 ± 6 nm, detection at 520 ± 5 nm) for 12 cycles (~17 min total) at 37°C. All dispensing was done using the BioTek MultiFlo FX. All screening plates contained positive-control wells (lacking CSR3) and nega- tive-control wells (25 nl dimethyl sulfoxide, vehicle), which were used as standards to calculate the percentage of inhibition (PI) of each compound (see equation above in Data analysis).
HTS assay results
The FRET-based assay was utilized in a primary screen of 6620 small molecules of diverse structure. Of these 6620 compounds, 109 (1.66%) had a PI >30% (for PI distribution of those 109 com- pounds, see Table 2). The 12 compounds with a PI value >90% had diverse structures, and no common scaffold was readily apparent (Fig. 4).
A dose-response assay was carried out with the top 109 compounds (concentration range, 1.25 nM to 50 μM). The data were analyzed with Breeze software, which generates dose-response curves and calculates the half-maximal inhibitory concentration (IC50) and drug-sensitivity score (DSS) for every compound. Con- sidering that IC50 alone cannot comprehensively evaluate drug sen- sitivity of dose-response model in HTS assay, DSS was developed by Yadav et al. (2014), Sci Rep, 4, 5193 as a systematic algorithmic solution that integrated five factors including IC50, the slope at IC50, minimum activity level, and top and bottom asymptotes of dose-response model. The DSS values ranged from 0 to 19.2. Table 3 and Figs. 5A to 5C present data for the top 3 compounds based on DSS values. The three structures differ (Fig. 4, starred com- pounds). In addition, of the 12 compounds with PI > 90% in the primary screen, 10 were among the top 20 most potent compounds as determined in the dose-response screen, indicating that the assay
was internally consistent even though a single CSR3 concentration was used.
Table 3 The top three most potent inhibitors of CSR3 as determined with the dose-response assay based on DSS values.
In summary, 109 CSR3 inhibitors were identified in the assays with a PI value of 30% as a threshold. By raising the threshold, one is able to effectively reduce the number of poten- tial false positives, and induce false negatives. The best three compounds (FIMM027745 (Cclccc(C)c(cl)C (=0)CSclncn[nH]1 according to the SMILES notation), FIMM072577 (Oclccc2c(cccc2cl)S([0- ]) (=0)=0), and FIMM031755 (Cclccc(ccl)[C@@H]1NC2CCC(cc2[C@@H]2C=CC[C@H]12)C(0)=0)), accord- ing to their DSS in the dose-response screening of CSR3, were all synthetic compounds. Moreover, all three compounds have not been reported as effective drugs according to Drugbank (https://www.drugbank.ca/). However, according to PubChem data- base, FIMM031755 (CID: 7114450) has been involved in an inhibitor screening assay for Cytokine/receptor Binary Complex of humans (4KC3_B) and was shown to be effective at micromolar concentra- tions. FIMM027745 (CID: 712810) has been used in several inhibitor screens, e.g. Hiv-1 Reverse Transcriptase and human heat shock 70 kDa protein, but was inactive in all studies.
EXAMPLE 4
Inhibitor validation assay in planta
Two compounds were tested at several concentrations (0.1 nM to 100 mM) on sweet potato plants co-infected with both SPCSV and SPFMV grown in medium. Preliminary results of the two compounds FIMM027749 (SMILES notation COclccc(cclOC)C (=0)CSclnnc[nH]1) and FIMMO72436 (SMILES notation CC (C)(C)nlcccc(C=NNC (N)=S)cl=S;
structure as follows:
with different PI val- ues (88% and 41%, respectively) showed that FIMM027749 was able to down-regulate virus accumulation of both SPCSV and SPFMV by 2-3 times, but without displaying typical dose-response curve (Fig. 6A, 6B). Compound FIMM072436, on the contrary, had a mild impact on SPFMV but no impact on SPCSV (Fig. 6A, 6B). Their effects on virus accumulation in plants were consistent with PI values ob- tained by in vitro assay. At the same time, plant height over time was estimated and both compounds did not have any effect on plant growth (see plant picture in Fig. 6C), suggesting that they did not interfere with plant development through unspecific interac- tion with host factors including endogenous RNase III.
The HTS assay could be used to identify inhibitors of various class 1 RNase III enzymes.
EXAMPLE 5
High-throughput screening strategy
Inhibitor identification for CSR3 was done in four phases (Fig. 7). In Phase one, CSR3 structure was modeled and virtual screening by Glide-docking was performed with 136,353 compounds targeting the active site of CSR3. In phase two, compound screening in laboratory was performed including a FRET-based HTS set up and two binding affinity assays using MST and SPR. Phase three was a validation assay in planta where the effects of inhibitors on viral accumulation were monitored using RT-qPCR and imaging-based meth- ods in sweet potato grown in medium or soil, respectively. Phase four was a posterior cluster study of the hits based on the com- pound structures.
Structural modeling and virtual screening
Amino acid sequence analysis showed that CSR3 was rather similar in size to RNase III from E.coli (EcR3), Aquifex aeolicus (AaR3) and Thermotoga maritima (TmR3) with 228, 226, 221 and 240 residues, respectively (Fig. 8A). All these proteins are proto- typical class 1 RNase III enzymes which have been well studied structurally and functionally. These enzymes are composed of an
endoND and a dsRBD connected by a flexible linker as represented by the CSR3 structure in Fig. 8B. The catalytic site is composed of four amino acids 40E, 44D, 126N, and 129E in CSR3 (Fig. 8A), while 107D in AaR3 corresponds to 126N in CSR3, see the superposed catalytic site of CSR3 and AaR3 (PDB 2NUG, 1.7 A) in Fig. 8B. These amino acids are essential for the catalytic activity of the enzyme. The side chains of these four amino acids are negatively charged or can be deprotonated allowing attraction of positively charged metals e.g. Mg2+, which further attract the negatively charged phosphates (-P04-) of dsRNA. The two-metal-ion mediated catalytic mechanism of RNase III has been described earlier. In our study, 136,353 compounds were Glide-docked into the catalytic site of CSR3, and 6,620 compounds were selected for further experimental testing according to their GlideScore rank order.
EXAMPLE 6
Protein preparation and HTS assay setup was performed as described in the previous Examples.
Homology modeling and virtual screening
EcR3, TmR3, and AaR3 from E.coli, Aquifex aeolicus, and Thermotoga maritima, respectively, were used for sequence align- ment with MAFFT (Katoh et al., 2002, Nucleic Acids Res.
30 (14):3059-3066). The model of CSR3 was built by I-TASSER. I- TASSER identify templates structure by LOMETS server, then select and score the templates of the highest significance in the thread- ing alignments which were used to simulate a pool of protein structure decoys. Finally, the top five models are identified ac- cording to pair-wise structure similarity using SPICKER program (Roy et al., 2010, Nature Protocols 5(4):725-738). In our study, the top identified template structures are PDB 1O0W, 5B16, 3C4T,
2EB1, 2A11, 2FFI, 302R, 2NUG, 1YYK, 4CE4. The highest-ranked model with I-TASSER c-score 0.56, TM-score 0.7910.09 was selected to further Glide-docking. The selected CSR3 model was processed with Protein Preparation Wizard of Schrodinger (Schrodinger releases 2016-4: LLC, New York, NY). Structures of 136,353 small molecules from the High Throughput Biomedicine Unit (HTB) of the Institute for Molecular Medicine Finland (FIMM) were prepared with LigPreg
function of Schrodinger with default setup. Active site residues of CSR3 (40E, 44D, 126N, and 129E) were selected as the center of Glide-Grid box, and docking was performed using SP and XP scoring modes employing OPLS3 force field under default settings (Friesner et al., 2004, J Med Chem 47(7):1739-1749; Friesner et al., 2006, J Med Chem 49(21):6177-6196). Based on ranking of GlideScore, 6,620 out of 136,353 compounds were selected to laboratory screening.
FRET-based screening in one concentration
6,620 compounds selected from Glide-docking were screened using the HTS assay described in the Examples above.
Compounds were screened at a final concentration of 10 mM on the 20 screening plates. All screening plates contained negative control reactions with CSR3 and DMSO (0.125%) and positive controls without CSR3. Their fluorescence trends were very different see Fig. 9. Slopes of the positive and negative controls were used to evaluate the suitability of the HTS with the parameter Z prime (Z') and its threshold is 0.5. The average Z' was 0.82 ± 0.04 for 20 screening plates, indicating the screening assay was techni- cally successful and results were quantitatively good for screen- ing. PI of all compounds was calculated with fluorescence slopes by considering controls (see data analysis). As a result, 109 compounds displaying PI >30% were selected for further testing, which contains 12 compounds had PI >90% (Fig. 10A).
Dose-response assay
The dose-response assay containing six concentrations (1.25 nM - 50 mM) was carried out in total three times, including two repeated tests with the selected 109 FIMM compounds and one test using newly ordered commercial compounds (99 of the 109 com- pounds; i.e., compounds prepared by a second supplier) (Fig. 10B). In these experiments, dose-response curve for each compound was generated using the PI values, and then half-maximal inhibitory concentration (IC50) and drug sensitivity score (DSS) were deter- mined according to Yadav et al. (2014), Sci Rep, 4, 5193. The DSS value was used to score the sensitivity of individual compounds. The results showed that DSS values varied between 0 and 22, taking threshold DSS >4, 41 compounds were selected for the next step in consideration of all three replicates in Fig. 10B (rectangle in the intersection of the circles).
EXAMPLE 7
Binding affinity assay using MST
Proteins were labeled using Red-Tris-NTA dye (NanoTemper, Miinchen, Germany) and resuspended in 50 μl of PBS buffer (137 mM NaCl, 2.7 mM KC1, 10 mM Na2HP04, 2 mM KH2P04, pH 7.4) with 0.05% Tween-20 to obtain 5 mM dye solution. Labelled-protein solution containing 500 nM proteins and 40 nM dye was prepared in PBS buffer with 2% DMSO for the assay. The 12 concentrations for each compound were obtained by 2-fold serial dilutions (400 mM — 2 mM). A peptide control was performed to discriminate binding-specific fluores- cence quenching from loss of fluorescence due to protein precipi- tation. Two independent experiments were carried out in Premium Coated Capillaries using MST power set on high (80%); LED power (pico red) set on 5% and on-Time 20 s. The dissociation constant (Kd) was determined using the MO.Affinity Analysis (NanoTemper).
Binding affinity assay was carried out with the 99 com- mercial compounds. Firstly, all 99 compounds were tested using MST. As a result, 36 compounds were selected as binders by taking into consideration four conditions: 1) raw fluorescence induced by compounds excluding their initial fluorescence, 2) signal/noise >5, 3) response amplitude >4, and 4) Kd lower than 200 mM (Fig 10C, the top right circle indicated as "MST").
Binding affinity assay using SPR
SPR was performed on a BiacoreTlOO instrument (GE Healthcare) using sensor S CM5 chip. Proteins (10 ng/μl) were immobilized using the standard amine coupling method according to manufactory instructor with immobilization buffer (10 mM NaAc pH=4), and final response units (RU) were 11,459. Proteins were purified as described above and stored in PBS buffer. Compounds of one concentration (100 mM) or 2-fold serial dilutions (3 mM — 200 mM) were flown over the sensor surface using PBS buffer with 0.01 M HEPES, 0.05% v/v P20, and 2% DMSO. compounds were tested from lower to higher concentration at 25°C, injection and dissociation were done at a flow rate of 30 μl/min for 60 s and 300 s, respec- tively. To eliminate bulk interference of DMSO, a solvent correc- tion of DMSO concentration ranging from 1.5% to 2.8% was tested
every 30 samples. SPR data were evaluated with the BiacoreTlOO Evaluation software 2.04.
Considering the results of HTS and MST, 56 compounds were screened using SPR with a concentration 100 mM. In a condition of relative response (RU) value >10, 42 compounds were further tested dosage with 12 concentrations (3 mM - 200 mM). 36 compounds were selected as good binders taking into consideration of steady-state affinity and the kinetics graphs of the dose-response assay (Fig 10C, the bottom circle indicated as "SPR"). To this point, gener- ally considering all laboratory screening steps HTS, MST, and SPR, 30 compounds were selected as potential inhibitors (Fig. 4C, cir- cled in the intersections of the three circles).
EXAMPLE 8
Plant material and growth conditions
Sweet potato (cultivar Huachano, CIP42006) were side graft-inoculated with both SPFMV (East African strain isolate Naml) and SPCSV-Ug (East African serotype 2) as described in Kreuze et al., 2008, Mol Plant Pathol 9(5):589-598 and Wang et al., 2008, J Virol Methods 154(1-2):135-145. Plantlets were propagated by taking single-node stem grown in culture medium (Wang et al., 2019, Plant Methods 15(1):116). Then plantlets with newly formed roots were transferred to glass tubes (18 x 150 mm) containing 10 ml of medium supplemented with either 50 mM of compound (diluted in DMSO), or only 0.1% DMSO as control. For plant experiments in soil, plantlets were transferred to pots (6 x 6 x 10 cm) filled with a mix out of 1/3 sand, 1/3 humus, and 1/3 washed soil. After one week, plants were treated by foliar spraying using either 10 mM compound (treatment) or water (control), twice a week over a month. All plants were grown at 22°C with 60% of humidity and a 16/8h light/dark photoperiod for 28 days in culture medium and 41 days in soil.
Virus accumulation assay with RT-qPCR
From plants grown in culture medium, leaf samples were collected and frozen in liquid nitrogen after 28 days. Total RNA was extracted using the Spectrum plant total RNA kit (Sigma-Al- drich). First-strand cDNA was synthesized using the Transcriptor
1st cDNA synthesis kit (Roche, Basel, Switzerland). Gene expres- sion was measured in a final 10 μl volume (containing 2 μl lOx diluted cDNA, 5 μl SYBR Green I Master Mix (Roche), and 2.5 mM primers) using the LightCycler 480 instrument II (Roche). All RT- qPCR experiments were conducted in triplicate on 3 biological rep- licates. Primer list can be found in Wang et al., 2019, Plant Methods 15(1):116. Relative gene expression was calculated using the classical 2~DD0T method since the efficiency of all primer pairs were close to 100% and showed less than 5% difference between them.
Inhibitor screening in sweet potato plants grown in cul- ture medium
Sweet potato plants co-infected with SPCSV and SPFMV grown on culture medium separately supplemented with 50 mM each compound were used to evaluate the effects of compounds on plants. In total 55 compounds were screened in planta considering the results of HTS and affinity assays. Their effects were monitored by plant height by imaging the plants once a week. 7 of 55 compounds showed stress symptoms, e.g. deformation, wilting, bleaching, dried leaf margins, or severe growth defects, possibly because of their toxicity to plants. Moreover, virus accumulation was quan- tified using the relative expression of their coat proteins as described in Kokkinos & Clark, 2006, Plant Dis. 90(6):783-788 and Kreuze et al., 2008, Mol Plant Pathol 9(5):589-598, and compounds' effects on accumulation was estimated by comparing treated and control plants.
13 and 20 compounds were selected according to reduction of SPCSV and SPFMV accumulation, respectively, with a threshold of 60% (Fig. 11A, the sections indicated as SPFMVCO.6 and SPCSVC0.6). Among them, five compounds showed for both viruses accumulation (Fig. 11A). Compared to control, their log2 fold change for both viruses showed in Fig. 11B. Specifically, SPCSV was reduced three times by two compounds (FIMM022230 (OC (=0)clccc(ccl)ClNc2c(Br)cccc2C2C=CCC12 according to the SMILES notation) and FIMM005536 (OC (=0)clccc2NC (C3CC=CC3c2cl)clcccccl)), two times by two compounds (FIMM051696 (OC (=0)clccc2NC (C3CC=CC3c2cl)clccc(F)ccl) and FIMM000096 (CN1CCC2=C3C1CC4=CC=C (0)C (=C4C3=CC=C2)0)), and one time by the compound FIMM031755
(Cclccc (ccl)[C@@H]1NC2CCC(cc2[C@@H]2C=CC[C@H]12)C (0)=0). SPFMV was reduced two times by three compounds (FIMM022230, FIMM005536, and FIMM051696) and one time by two compounds (FIMM000096 and FIMM031755). Overall, the five compounds reduced both SPCSV and SPFMV accumulation without any phytotoxicity effects in sweet po- tato plants, see experimental plant pictures in Fig. 11C.
EXAMPLE 9
High-throughput plant phenotyping platform
Finnish National Plant Phenotyping Infrastructure (NaPPI) was applied to monitor plant virus disease symptoms. Measurements were obtained twice a week over 40 days. An RGB camera (IDS Imaging Development Systems GmbH, Obersulm, Germany) was used for the top- view imaging of plants, and plant surface area was delineated using software Morpho Analysis 1.0.5.1 (PSI, Brno, Czech Republic). A fluorescence camera (400-1000 nm) with a pulse amplitude modula- tion system (PSI) was used for ChlF imaging, which was analyzed with the FluorCam 7.0 software. Specifically, shutter and sensi- tivity were adjusted for the plant material to 33.33 ys and 5%, respectively. 4>PSII parameter and its false-color images were gen- erated by the Plantscreen Data Analyzer software (PSI). Four com- pounds were tested in the phenotyping platform in two independent batches including five biological replicates each.
Inhibitor validation in planta using sweet potato plants grown in soil
The effect of four out of the five most promising com- pounds (FIMM000096, FIMM031755, FIMM022230, and FIMM005536) were monitored on co-infected sweet potatoes grown in soil using a plant phenotyping platform. At the physiological level, the quantum yield of PSII (4>PSII) was used to monitor the effect of the com- pounds on photosynthetic performance. 4>PSII, indicating the pro- portion of light used by chlorophyll associated with PSII in sweet potato, is an efficient estimator of viral effects in sweet pota- toes (Wang et al., 2019, Plant Methods 15(1):116). Herein, sweet potato plants treated with all four compounds displayed a signif- icant increase of 4>PSII values compared to controls reflecting improved photosynthetic performance (Fig. 11D, Dunnett's test).
The effects on 4>PSII were confirmed by a significant reduction of SPFMV accumulation (Fig. 11F, Dunnett's test). At the morphologi- cal level, after one month of compound treatment, none of the treated plants showed signs of plant stress as shown by visible light (RGB) imaging in Fig. 11 E. Taken together, the results demon- strated that the four compounds had a positive effect on photo- synthesis performance reflecting the improved condition of co- infected sweet potatoes.
EXAMPLE 10
In summary
To diminish the effect of synergistic viral infection of sweet potato causing severe crop loss, inhibitors targeting CSR3, an RNA silencing suppressor, were screened. Following the virtual screening, FRET-based HTS was performed to effectively quantify changes of CSR3 activity caused by compounds. Subsequently, bind- ing affinity between CSR3 and compounds was monitored by MST and SPR methods. Finally, the effect of the selected compounds on viral accumulation, photosynthetic performance, and plant growth was further validated in planta by RT-qPCR and imaging-based plant phenotyping. As a result, four compounds were identified as ef- fective inhibitors of CSR3.
At present, antiviral strategies in plants are based on either breeding virus-resistant cultivars or targeting viruses to prevent viral replication and spreading. Another effective strat- egy, widely used for animal viruses and often forsaken in plants, is antiviral drug identification by targeting different stages of the viral life-cycle. Conventionally, most virus control strate- gies are applied in pre-infected plants, which emphasizes the need for the development of alternative anti-viral strategies in post- infected plants such as antiviral inhibitor identification. HTS of small molecules may be used for antiviral inhibitor discovery. Increasingly, HTS targeting viral RNA silencing suppressors has become a potential approach to control virus diseases. The present experiments focused on the identification of inhibitors for an RNA silencing suppressor CSR3 encoded by SPCSV. Many inhibitors of RNase H have been found, e.g. N-hydroxyimide (2-hydroxy-4H-
isoquinoline-1,3dione), F3284-8495, and a series of N-hydroxyimide compounds. However, RNase H enzymes are functionally very differ- ent from class 1 RNase Ills, as RNase H enzymes hydrolyze the RNA strands of DNA/RNA duplexes during reverse transcription. As ex- pected, inhibitors of RNase H were not docking well to CSR3 in silico.
Glide-docking, used in the present examples, is a com- plete and hybrid method for searching potential docking poses with high accuracy. However, because the structural model of CSR3 was used in silico, a relatively large number (6,620) of small mole- cules were screened in laboratory. In addition, targeting of highly conserved amino acid residues in the active site of CSR3 could reduce resistance breaking, which is an important feature in the development of sustainable antiviral strategies. Primary labora- tory screening was carried out using a FRET-based HTS built-up in the lab. FRET-based methods have pros, such as sensitivity and efficiency, but also cons which are likely to produce false-posi- tive and false-negative results. In the present examples, false- positive results could be obtained in two conditions: 1) compounds could directly interact with the substrate instead of CSR3 to prevent labelled-siRNA cleavage; 2) compounds could exhibit in- trinsic fluorescence with similar absorption and emission spectra as the fluorophore reporter. On the other hand, false-negative findings would be obtained if compounds quench the reporter fluor- ophore.
To sort out false-positive or -negative results from FRET-based primary screening, subsequent validations may be ap- plied. Two complementary methods, MST and SPR, were used to di- rectly measure the binding affinity between the CSR3 and compounds in our study. MST records the motion of molecules in microscopic temperature gradients detecting changes in hydration shell, charge, or size. However, since MST is a fluorescence-based method, and thus susceptible to disturbance by intrinsically fluorescent compounds, self-fluorescent compounds with similar fluorescent properties cannot be analyzed by this approach. In the complemen- tary SPR method, the interaction between protein and compounds is measured by monitoring small changes of an optical reflective index at the sensor surface. However, some compounds cannot properly
dissociate from the sensor, which will affect the assay of the next analyte. Thus, such compounds were identified in the prelim- inary one concentration test in SPR and excluded from further dose- response tests.
To exclude the inhibitor candidates could interfere with endogenous RNase III and impact to plant growth, validation tests in planta were carried out and most of the compounds (48 of 55) were not toxic to plants. Considering their effect on virus accu- mulation in plants grown in medium, five best inhibitors were identified. Four of them could be provided in a sufficient amount to confirm in plants grown in soil and all the four compounds showed significant improvement in photosynthesis performance of co-infected sweet potato. From the structure side, the best five candidates were clustered in two classes (Fig . 12 ) , which could be used to further characterize existing molecules to identify opti- mal candidates by studying structure-activity relationships. Among the five inhibitors, class 1 compound FIMM000096 has been approved as a powerful emetic and also been used in the treatment of par- kinsonism, but with adverse effects https://www.drug- bank.ca/drugs/DB00714 . The other four compounds, belonging to class 2, appear to have not been reported either in the Drugbank database or for the treatment of virus diseases. But they do have been included in inhibitor screening for human enzymes or bacterial proteins according to PubChem database with FIMM022230 (CID: 2948389), FIMM031755 (CID: 7114450), FIMM005536 (CID: 2857906), FIMM051696 (CID: 4240943). They all were inactive in the studies except FIMM031755, showing activity when target to chain B, cyto- kine/receptor binary complex of human. The data described herein showed structure and DSS value of class 2 compounds were rather similar, but different Kd values of binding affinity were obtained, which could be plausible if the configuration of the binding site is different. In addition, the class 1 and other three class 2 compounds were confirmed both in plants grown in medium and soil.
The five compounds identified in laboratory experiments and validated in plants cluster hierarchically into two classes. FIMM000096 (SMILES notation CN1CCC2=C3C1CC4=CC=C (0)C (=C4C3=CC=C2)0) represents class 1 while the other four compounds (FIMM5536 [SMILES notation
OC (=0)clccc2NC (C3CC=CC3c2cl)clcccccl], FIMM031755 [SMILES nota- tion Cclccc(ccl) [C@@H]1NC2CCC(cc2[C@@H]2C=CC[C@H]12)C(0)=0], FIMMO51696 [SMILES notation OC (=0)clccc2NC (C3CC=CC3c2cl)clccc(F)ccl], and FIMM022230 [SMILES notation OC (=0)clccc(ccl)ClNc2c(Br)cccc2C2C=CCC12]) belong to class 2 with highly similar structures (Fig. 12). Fig. 12 also summarizes results from the FRET-based HTS, binding affinity as- says (MST or SPR) and in planta validation results from sweetpota- toes grown in culture medium. With FRET-based HTS, the compounds had similar DSS and IC50 values ranging from 12.4 to 15.9, and 1.27 to 2.9 mM, respectively. Their diminish of viruses accumula- tion in plants ranging from -0.77 to -3.56. However, the Kd values from binding affinity experiments varied between 0.69 mM to 3.44 mM among these compounds. The hit rate was 4.86% in Glide-docking in silico, 1.69% in FRET-based HTS, 36.4% in MST, 75% in SPR and 18.2% in viral screening in plants. Considering all screening steps, four compounds (hit rate 0.0037%) were identified as in- hibitors of CSR3.
EXAMPLE 11
Further compounds were screened based on the compounds found in previous Examples. The most potent inhibitors found are shown in Table 4.
It is obvious to a person skilled in the art that with the advancement of technology, the basic idea may be implemented in various ways. The embodiments are thus not limited to the examples described above; instead, they may vary within the scope of the claims.
The embodiments described hereinbefore may be used in any combination with each other. Several of the embodiments may be combined together to form a further embodiment. A method, a product, or a use, disclosed herein, may comprise at least one of the embodiments described hereinbefore. It will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments. The embodiments are not limited to those that solve any or all of the stated problems or those that have any or all of the stated benefits and advantages. It will further be understood that reference to 'an' item refers to one or more of those items. The term "comprising" is used in this specification to mean including the feature(s) or act(s) followed thereafter, without excluding the presence of one or more additional features or acts.
Claims
1. A composition comprising a compound represented by any one of formulas I to IV, or a salt thereof:
wherein Ri is H, Cl, F, Br, I, methyl, CF3, COOH, COO-R7, CONHR7, acetyl, alkyl, or aryl;
R2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF3, COO-R7, acetyl, alkyl, or aryl;
R3 is H, Cl, F, Br, I, methyl, ethyl, CF3, COOH, COO-R7, acetyl, SO2NHR7, CONHR7, alkyl, or aryl;
R4 is Br, H, CF3, COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, acetyl, or COO-R7;
R5 is H, acetyl, methyl, ethyl, alkyl, or aryl;
R6 is acetyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, cy- clohexenyl, phenyl, naphthyl, pyridyl, COOH, COO-R7, halogen, meth- oxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, H, or a C6 to C8 aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, -0(C0)R7, methyl, ethyl, propyl, isopropyl, n-bu- tyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclo- hexyl, halogen, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, hydroxyl, nitro, or H; wherein each R7 is independently -CH2CH2CH2COOH, CH2CH2COOH, -CH2COOH, alkyl, aryl, or a C6 to Cs aromatic ring optionally substituted with one or more substituents selected in- dependently from the group consisting of -COOH, -CH2-COOH, -CH2- CH2-COOH, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichlorome- thyl, hydroxyl, nitro, or H; and
wherein each alkyl and/or aryl is optionally substituted;
wherein
R8 to R12 are each independently selected from alkyl, methoxy, ethoxy, acetamido, phenyl, H, and halogen;
R14 and R17 are each independently selected from H, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H;
R15 and R16 are each independently selected from H, N¾, methyl, lower alkyl, and phenyl optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, Cl, F, Br, I, methoxy, ethoxy, trifluoromethyl, tribromomethyl, tri- chloromethyl, hydroxyl, nitro, or H; and wherein each alkyl is optionally substituted;
Formula III
wherein
Ri8 is H or lower alkyl;
Rig,R20, R22, R23, R24, and R25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy, ethoxy, and
NR37 ;
R26 to R30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy;
R37 is H, methyl, lower alkyl, phenyl, -(CO)CH2CH2CH2CH2CH3, - (CO)CH2CH2CH2CH3, -(CO)CH2CH2CH3, -(CO)CH2CH3, or -(CO)CH3; and wherein each alkyl is optionally substituted;
wherein R31 is H, methyl, ethyl, or lower alkyl;
R32, R33, R34, R35, and R36 are each independently selected from H, OH, methoxy, and ethoxy; and wherein each alkyl is optionally substituted.
2. The composition according to claim 1, wherein the com- position comprises a compound represented by any one of formulas I to IV, or a salt thereof:
Formula I
wherein R1 is H, Cl, F, Br, I, methyl, CF3, COOH, COO-R7, alkyl, or aryl;
R2 is H, COOH, Cl, F, Br, I, methyl, ethyl, CF3, COO-R7, alkyl, or aryl;
R3 is H, Cl, F, Br, I, methyl, ethyl, CF3, COOH, COO-R7, alkyl, or aryl;
R4 is Br, H, CF3, COOH, Cl, F, I, methyl, ethyl, alkyl, aryl, or COO-R7;
R5 is H, methyl, ethyl, alkyl, or aryl;
R6 is methyl, ethyl, propyl, isopropyl, n-butyl, sec- butyl, tert-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, phenyl, COOH, COO-R7, halogen, methoxy, ethoxy, trifluoromethyl, tribro- momethyl, trichloromethyl, H, or a C6 to Ce aromatic ring optionally substituted with one or more substituents selected independently from the group consisting of -COOH, -COO-R7, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclo- pentyl, cyclohexyl, halogen, methoxy, ethoxy, trifluoromethyl, tribromomethyl, trichloromethyl, hydroxyl, or H; wherein each R7 is independently alkyl or aryl; and wherein each alkyl and/or aryl is optionally substituted;
Formula II wherein
R14,and R17 are each independently selected from H, methyl, and lower alkyl;
R15 and R16 are each independently selected from H, NH2, methyl and lower alkyl; and
wherein each alkyl is optionally substituted;
Formula III wherein
R18 is H or lower alkyl;
Rig,R20, R22, R23, R24, and R25 are each independently selected from H, OH, halogen, methyl, lower alkyl, methoxy and ethoxy; R21 is H, OH, halogen, methyl, lower alkyl, methoxy, eth- oxy, herein
R26 to R30 are each independently selected from H, OH, halogen, lower alkyl, methoxy and ethoxy; and wherein each alkyl is optionally substituted;
wherein R31 is H, methyl, ethyl, or lower alkyl;
R32, R33, R34, R35, and R36 are each independently selected from H, OH, methoxy, and ethoxy; and wherein each alkyl is optionally substituted.
4. The composition according to any one of claims 1 - 3, wherein the composition is a crop protection composition.
5. The composition according to any one of claims 1 - 4, wherein the composition further comprises one or more additives, vehicles, formulation auxiliaries, extenders, fillers and/or sur- face-active agents; and/or at least one pesticidally active sub- stance.
6. A method for treating a plant curatively and/or pre- ventively against at least one plant pathogenic virus and/or against sweet potato virus disease (SPVD), wherein the method com- prises applying a compound represented by any one of formulas I to IV as defined in claim 1 or 2 or a salt thereof, or a compound defined in claim 3 or a salt thereof, or a composition according to any one of claims 1 - 5, to the plant or a part thereof, a cutting for growing the plant, and/or a tuberous root of the plant,
or to soil and/or to substrate in which the plant is growing or in which it is desired to grow.
7. A compound, or a composition comprising a compound, wherein the compound is capable of inhibiting RNase III encoded by Sweet potato chlorotic stunt virus (SPCSV) or identified as capable of inhibiting RNase III encoded by SPCSV by a binding assay and/or as capable of reducing the accumulation of at least one plant pathogenic virus in a plant.
8. The compound or composition according to claim 7, wherein the binding assay is an assay based on fluorescence reso- nance energy transfer (FRET), microscale thermophoresis (MST) and/or surface plasmon resonance (SPR).
9. The use of a compound represented by any one of for- mulas I to IV as defined in claim 1 or 2 or a salt thereof, or of a compound defined in claim 3 or a salt thereof, or of a composition according to any one of claims 1 - 5, for treating a plant cura- tively and/or preventively against symptoms caused by at least one plant pathogenic virus and/or against sweet potato virus disease (SPVD).
10. The use of a compound represented by any one of for- mulas I to IV as defined in claim 1 or 2 or a salt thereof, or of a compound defined in claim 3 or a salt thereof, or of a composition according to any one of claims 1 - 5 for inhibiting RNase III encoded by SPCSV and/or for reducing the accumulation of at least one plant pathogenic virus in a plant.
12. The method according to claim 6 or 11, or the use according to any one of claims 9 - 11, wherein the at least one plant pathogenic virus comprises or is SPCSV, optionally syner- gistic with at least one other plant pathogenic virus.
13. The method according to claim 6 or 11 - 12 or the use according to any one of claims 9 - 12, wherein SPCSV is synergistic with one or more of Potyviruses (SPFMV, SPLV and/or SPMSV), Cu- cumoviruses (CMV), Ipomoviruses (SPMMV), Carlaviruses (SPCFV and/or C-6 virus), and/or Cavemoviruses (SPVCV and/or SPCV).
14. The method according to claim 6 or 11 - 13 or the use according to any one of claims 9 - 13, wherein SPCSV is synergistic with SPFMV and optionally with one or more of other Potyviruses (SPLV and/or SPMSV), Cucumoviruses (CMV), Ipomoviruses (SPMMV), Carlaviruses (SPCFV and/or C-6 virus), and/or Cavemoviruses (SPVCV and/or SPCV).
15. The method according to claim 6 or 11 - 14 or the use according to any one of claims 9 - 14, wherein the plant is a plant of Ipomoea spp.
16. The method according to claim 6 or 11 - 15 or the use according to any one of claims 9 - 15, wherein the plant is sweet potato.
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Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030087926A1 (en) * | 2000-02-07 | 2003-05-08 | Gruenenthal Gmbh | Substituted 1,2,3,4-tetrahydroquinoline-2-carboxylic acid derivatives |
US20080214537A1 (en) * | 2007-03-02 | 2008-09-04 | Cara Therapeutics, Inc. | Bridged phenanthridines |
WO2009076665A1 (en) * | 2007-12-13 | 2009-06-18 | Indiana University Research And Technology Corporation | Materials and methods for inhibiting mammalian s-nitrosoglutathione reductase |
CN102150670A (en) * | 2006-11-23 | 2011-08-17 | 南开大学 | Application of phenanthroindolizidine and phenanthroquinolizidine derivatives and salts thereof in pesticides |
WO2013123081A2 (en) * | 2012-02-14 | 2013-08-22 | Children's Hospital Medical Center | Use of small molecule inhibitors targeting the interaction between rac gtpase and p67 (phox) |
WO2013149997A1 (en) * | 2012-04-02 | 2013-10-10 | Almirall, S.A. | Substituted tricyclic compounds with activity towards ep1 receptors |
US20150018326A1 (en) * | 2013-07-12 | 2015-01-15 | Children's Hospital Medical Center | Compositions and methods for inhibiting norovirus infection |
WO2016077793A1 (en) * | 2014-11-14 | 2016-05-19 | Children's Hospital Medical Center | Sos1 inhibitors for cancer treatment |
EP3091015A1 (en) * | 2013-12-30 | 2016-11-09 | Nankai University | Beta-carboline, dihydro-beta-carboline and tetrahydro-beta-carboline alkaloid derivatives and method for preparing same and use in aspects of preventing and treating plant viruses, fungicides and insecticides |
WO2017050262A1 (en) * | 2015-09-25 | 2017-03-30 | 南开大学 | Phenanthroindolizidine alkaloid quaternary ammonium salt derivatives, and preparation thereof and application thereof in resistance to plant viruses |
CN109096257A (en) * | 2018-08-09 | 2018-12-28 | 山东博苑医药化学有限公司 | The application of Meridianin Alkaloid and its derivative in prevention and treatment plant virus germ disease |
CN109516978A (en) * | 2019-01-28 | 2019-03-26 | 河北工业大学 | Giantreed alkali derivant and its preparation method and application |
CN110483405A (en) * | 2018-05-14 | 2019-11-22 | 南开大学 | Kealiinine analog derivative and its preparation and the application in Antiphytoviral and germ |
CN110615792A (en) * | 2019-11-01 | 2019-12-27 | 河北工业大学 | 2-benzylthio-5-methyl-4, 7-dihydro- [1,2,4] triazolo [1,5-a ] pyrimidine derivatives |
CN110759896A (en) * | 2018-07-26 | 2020-02-07 | 南开大学 | Piperazine dione acylhydrazone derivative, preparation method thereof and application of piperazine dione acylhydrazone derivative in plant virus prevention and control, sterilization and disinsection |
CN110759913A (en) * | 2018-07-26 | 2020-02-07 | 南开大学 | Indolylhydrazone derivatives, preparation method thereof and application thereof in plant virus prevention and control, sterilization and disinsection |
-
2021
- 2021-04-19 WO PCT/FI2021/050285 patent/WO2021209689A1/en active Application Filing
Patent Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030087926A1 (en) * | 2000-02-07 | 2003-05-08 | Gruenenthal Gmbh | Substituted 1,2,3,4-tetrahydroquinoline-2-carboxylic acid derivatives |
CN102150670A (en) * | 2006-11-23 | 2011-08-17 | 南开大学 | Application of phenanthroindolizidine and phenanthroquinolizidine derivatives and salts thereof in pesticides |
US20080214537A1 (en) * | 2007-03-02 | 2008-09-04 | Cara Therapeutics, Inc. | Bridged phenanthridines |
WO2009076665A1 (en) * | 2007-12-13 | 2009-06-18 | Indiana University Research And Technology Corporation | Materials and methods for inhibiting mammalian s-nitrosoglutathione reductase |
WO2013123081A2 (en) * | 2012-02-14 | 2013-08-22 | Children's Hospital Medical Center | Use of small molecule inhibitors targeting the interaction between rac gtpase and p67 (phox) |
WO2013149997A1 (en) * | 2012-04-02 | 2013-10-10 | Almirall, S.A. | Substituted tricyclic compounds with activity towards ep1 receptors |
US20150018326A1 (en) * | 2013-07-12 | 2015-01-15 | Children's Hospital Medical Center | Compositions and methods for inhibiting norovirus infection |
EP3091015A1 (en) * | 2013-12-30 | 2016-11-09 | Nankai University | Beta-carboline, dihydro-beta-carboline and tetrahydro-beta-carboline alkaloid derivatives and method for preparing same and use in aspects of preventing and treating plant viruses, fungicides and insecticides |
WO2016077793A1 (en) * | 2014-11-14 | 2016-05-19 | Children's Hospital Medical Center | Sos1 inhibitors for cancer treatment |
WO2017050262A1 (en) * | 2015-09-25 | 2017-03-30 | 南开大学 | Phenanthroindolizidine alkaloid quaternary ammonium salt derivatives, and preparation thereof and application thereof in resistance to plant viruses |
CN110483405A (en) * | 2018-05-14 | 2019-11-22 | 南开大学 | Kealiinine analog derivative and its preparation and the application in Antiphytoviral and germ |
CN110759896A (en) * | 2018-07-26 | 2020-02-07 | 南开大学 | Piperazine dione acylhydrazone derivative, preparation method thereof and application of piperazine dione acylhydrazone derivative in plant virus prevention and control, sterilization and disinsection |
CN110759913A (en) * | 2018-07-26 | 2020-02-07 | 南开大学 | Indolylhydrazone derivatives, preparation method thereof and application thereof in plant virus prevention and control, sterilization and disinsection |
CN109096257A (en) * | 2018-08-09 | 2018-12-28 | 山东博苑医药化学有限公司 | The application of Meridianin Alkaloid and its derivative in prevention and treatment plant virus germ disease |
CN109516978A (en) * | 2019-01-28 | 2019-03-26 | 河北工业大学 | Giantreed alkali derivant and its preparation method and application |
CN110615792A (en) * | 2019-11-01 | 2019-12-27 | 河北工业大学 | 2-benzylthio-5-methyl-4, 7-dihydro- [1,2,4] triazolo [1,5-a ] pyrimidine derivatives |
Non-Patent Citations (34)
Title |
---|
"GenBank", Database accession no. ADQ42569.1 |
BABU G ET AL: "Imino Diels-Alder Reactions Catalyzed by Indium Trichloride (InCl"3). Facile Synthesis of Quinoline and Phenanthridinone Derivatives", TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM , NL, vol. 38, no. 28, 14 July 1997 (1997-07-14), pages 5025 - 5026, XP004081285, ISSN: 0040-4039, DOI: 10.1016/S0040-4039(97)01060-5 * |
BABU G ET AL: "Indium Trichloride (InCl"3)Catalyzed Imino Diels-Alder Reactions: An Efficient Synthesis of Cyclopentaquinolines, Azabicyclooctanones and Azabicyclononanones", TETRAHEDRON, ELSEVIER SIENCE PUBLISHERS, AMSTERDAM, NL, vol. 54, no. 8, 19 February 1998 (1998-02-19), pages 1627 - 1638, XP004107445, ISSN: 0040-4020, DOI: 10.1016/S0040-4020(97)10370-2 * |
BYUNG-HOON LEE ET AL: "Enhancement of proteasome activity by a small-molecule inhibitor of USP14", NATURE, vol. 467, no. 7312, 9 September 2010 (2010-09-09), London, pages 179 - 184, XP055467744, ISSN: 0028-0836, DOI: 10.1038/nature09299 * |
BYUNG-HOON LEE ET AL: "Enhancement of proteasome activity by a small-molecule inhibitor of USP14", NATURE, vol. 467, no. 7312, 9 September 2010 (2010-09-09), pages 179 - 184, XP055123891, ISSN: 0028-0836, DOI: 10.1038/nature09299 * |
CARLINO LUCA ET AL: "Structure-Activity Relationships of Hexahydrocyclopenta[c]quinoline Derivatives as Allosteric Inhibitors of CDK2 and EGFR", CHEMMEDCHEM, vol. 13, no. 24, 1 January 2018 (2018-01-01), pages 2627 - 2634, XP055809848 * |
CHRISTODOULOU MICHAEL S ET AL: "Probing an Allosteric Pocket of CDK2 with SMall Molecules", CHEMMEDCHEM, vol. 12, no. 1, 1 January 2017 (2017-01-01), pages 33 - 41, XP055809852 * |
CIUSTEA MIHAI ET AL: "10.1021/jm800366g Identification of Non-Nucleoside DNA Synthesis Inhibitors of Vaccinia Virus by High-Throughput Screening - Supporting information", JOURNAL OF MEDICINAL CHEMISTRY, vol. 51, 1 January 2008 (2008-01-01), pages S1 - S10, XP055809926 * |
DUSICA VIDOVI ET AL: "Supporting Information: A Combined Ligand-and Structure-Based Virtual Screening Protocol Identifies Submicromolar PPARg Partial Agonists", 3 January 2011 (2011-01-03), XP055203589, Retrieved from the Internet <URL:http://onlinelibrary.wiley.com/store/10.1002/cmdc.201000428/asset/supinfo/cmdc_201000428_sm_miscellaneous_information.pdf?v=1&s=cc19b2737f247f8146ba818aa54a367a6ae8dd52> [retrieved on 20150721] * |
FRIESNER ET AL., J MED CHEM, vol. 47, no. 7, 2004, pages 1739 - 1749 |
FRIESNER ET AL., J MED CHEM, vol. 49, no. 21, 2006, pages 6177 - 6196 |
GORE V K ET AL: "Structure-activity relationship (SAR) investigations of tetrahydroquinolines as BKCa agonists", BIORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM , NL, vol. 20, no. 12, 15 June 2010 (2010-06-15), pages 3573 - 3578, XP027351206, ISSN: 0960-894X, [retrieved on 20100520] * |
JOHNSON STEVEN M ET AL: "A biochemical screen for GroEL/GroES inhibitors- Supporting Information", BIOORGANIC AND MEDICINAL CHEMISTRY LETTERS, 1 January 2014 (2014-01-01), pages 786 - 789, XP055809901, Retrieved from the Internet <URL:https://ars.els-cdn.com/content/image/1-s2.0-S0960894X13014844-mmc1.docx> * |
JOHNSON STEVEN M ET AL: "A biochemical screen for GroEL/GroES inhibitors", BIORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM , NL, vol. 24, no. 3, 2 January 2014 (2014-01-02), pages 786 - 789, XP028819909, ISSN: 0960-894X, DOI: 10.1016/J.BMCL.2013.12.100 * |
KATOH ET AL., NUCLEIC ACIDS RES., vol. 30, no. 14, 2002, pages 3059 - 3066 |
KISELYOV ALEXANDER S ET AL: "Solid Support Synthesis of Tetrahydroquinolines via the Grieco Three Component Condensation", TETRAHEDRON LETTERS, vol. 38, no. 35, 1997, pages 6163 - 6166, XP085609606, ISSN: 0040-4039, DOI: 10.1016/S0040-4039(97)01425-1 * |
KOKKINOSCLARK, PLANT DIS., vol. 90, no. 6, 2006, pages 783 - 788 |
KOPRA KARI ET AL: "High-Throughput Dual Screening Method for Ras Activities and Inhibitors", ANALYTICAL CHEMISTRY, vol. 89, no. 8, 29 March 2017 (2017-03-29), US, pages 4508 - 4516, XP055790220, ISSN: 0003-2700, Retrieved from the Internet <URL:https://pubs.acs.org/doi/pdf/10.1021/acs.analchem.6b04904> DOI: 10.1021/acs.analchem.6b04904 * |
KREUZE ET AL., J VIROL, vol. 79, 2005, pages 7227 - 7238 |
KREUZE ET AL., MOL PLANT PATHOL, vol. 9, no. 5, 2008, pages 589 - 598 |
MACCARI ROSANNA ET AL: "Low Molecular Weight Phosphotyrosine Protein Phosphatases as Emerging Targets for the Design of Novel Therapeutic Agents", JOURNAL OF MEDICINAL CHEMISTRY, vol. 55, no. 1, 12 January 2012 (2012-01-12), US, pages 2 - 22, XP055809860, ISSN: 0022-2623, Retrieved from the Internet <URL:https://pubs.acs.org/doi/pdf/10.1021/jm200607g> DOI: 10.1021/jm200607g * |
MIHAI CIUSTEA ET AL: "Identification of Non-Nucleoside DNA Synthesis Inhibitors of Vaccinia Virus by High-Throughput Screening", JOURNAL OF MEDICINAL CHEMISTRY, vol. 51, no. 20, 23 October 2008 (2008-10-23), pages 6563 - 6570, XP055203642, ISSN: 0022-2623, DOI: 10.1021/jm800366g * |
NINO PATRICIA ET AL: "ChemInform Abstract: Efficient Three-Component Synthesis of Diversely Substituted Tetrahydro-1H-cyclopenta[c]quinolines.", CHEMINFORM, vol. 55B, no. 7, 1 December 2016 (2016-12-01), pages 854 - 881, XP055809853, ISSN: 0931-7597, Retrieved from the Internet <URL:http://nopr.niscair.res.in/bitstream/123456789/35022/1/IJCB%2055B(7)%20854-881.pdf> DOI: 10.1002/chin.201652319 * |
PONTE CRISTIANO G. ET AL: "Selective, Direct Activation of High-Conductance, Calcium-Activated Potassium Channels Causes Smooth Muscle Relaxation", MOLECULAR PHARMACOLOGY, vol. 81, no. 4, 16 April 2012 (2012-04-16), US, pages 567 - 577, XP055809861, ISSN: 0026-895X, Retrieved from the Internet <URL:http://dx.doi.org/10.1124/mol.111.075853> DOI: 10.1124/mol.111.075853 * |
RITZ ET AL., PLOS ONE, vol. 10, 2015, pages e0146021 |
ROY ET AL., NATURE PROTOCOLS, vol. 5, no. 4, pages 725 - 738 |
VIDAL ET AL: "Structure-based discovery of new small molecule inhibitors of low molecular weight protein tyrosine phosphatase", EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 42, no. 8, 23 July 2007 (2007-07-23), pages 1102 - 1108, XP022165144, ISSN: 0223-5234, DOI: 10.1016/J.EJMECH.2007.01.017 * |
VIDOVIC DUSICA ET AL: "A Combined Ligand- and Structure-Based Virtual Screening Portocol Identifies Submicromolar PPARgPartial Agonists", CHEMMEDCHEM, vol. 6, no. 1, 1 January 2011 (2011-01-01), pages 94 - 103, XP055809869 * |
WANG ET AL., ANNALS OF APPLIED BIOLOGY, vol. 154, no. 3, 2009, pages 351 - 363 |
WANG ET AL., J VIROL METHODS, vol. 154, no. 1-2, 2008, pages 135 - 145 |
WANG ET AL., PLANT METHODS, vol. 15, no. 1, 2019, pages 116 |
YADAV ET AL., SCI REP, vol. 4, 2014, pages 5193 |
YADAV ET AL., SCI. REP., vol. 4, 2014, pages 5193 |
ZHANG ET AL., J BIOMOL SCREEN, vol. 4, 1999, pages 67 - 73 |
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