The control image in this case had brighter YFP fluorescence, for unknown reasons.(TIF) ppat.1007315.s016.tif (4.1M) GUID:?249E9D68-EA4B-49F5-A4F8-FA415EB10C0B S8 Fig: Sequences of CPSF3 from (TREU927), and sequence is present because of a gap in the genome assembly.(PDF) ppat.1007315.s017.pdf (723K) GUID:?5A535817-3B10-4D72-AD9A-F1C75B568E33 S9 Fig: Domain annotations, residues linked to resistance mutations in and mapped onto a structural model of sequence. and mildly resistant cells with AN7973 for 5h. For a detailed legend see the top sheet.(XLSX) ppat.1007315.s005.xlsx (30K) GUID:?DF68CD11-FBF0-4822-BA0E-DBC724314363 S6 Table: Levels of chosen metabolites after treatment with different compounds. For a detailed legend see the top sheet.(XLSX) ppat.1007315.s006.xlsx (48K) GUID:?6569E9F0-4F03-450A-BED8-BA2E6E2A297F S7 Table: Metabolomic effects from treating trypanosomes with Actinomycin D or AN5568 for 5h. (XLSX) ppat.1007315.s007.xlsx (256K) GUID:?C1684CBB-4A8D-48F3-8CA8-EBA72C459201 S8 Table: EC50s of various benzoxaboroles in cells with and without additional CPSF3 expression. (XLSX) ppat.1007315.s008.xlsx (10K) GUID:?C76785C2-C31E-4A05-9550-0B9680309558 S9 Table: Global validation of homology models. Details are in the Table.(XLSX) ppat.1007315.s009.xlsx (10K) GUID:?F5677B43-FB27-49CA-A91A-F67DF96BA016 S1 Fig: Statistical analysis of results in Fig 3. A: Results for an initial experiment in which the level of Y structure was quantified relative to in the presence and absence of benzoxaboroles. (PDF) ppat.1007315.s011.pdf (408K) GUID:?46DAE9FC-6E9C-4125-836D-413BA199EF44 S3 Fig: Time-to-kill assays for benzoxaboroleslike VTP-27999 S1 Fig but with more compounds and without the untreated control. (PDF) ppat.1007315.s012.pdf (77K) GUID:?A0873AD0-75AE-403F-94FA-70C33B603A40 S4 Fig: Effects of benzoxaboroles on Y structure levels. A. Time course of effects of AN7973, AN3967, AN5827 and AN11736 in bloodstream forms (Experiment 2 in Fig 7). Sinefungin treatment was for 30 min.B, C: effect of AN7973 in procyclic forms. (PDF) ppat.1007315.s013.pdf (377K) GUID:?066C3D3A-7295-4810-9CAB-5FD36B72BE59 S5 Fig: Results for three separate experiments (A, B and C) in which the location of YFP-DHH1 was analysed. The average percentages of cells with clear (dark blue) or possible (pale blue) peri-nuclear granules are plotted as bars, with the average numbers of large granules (at least 4 contiguous pixels at maximum intensity) anywhere in the cell displayed as black spots. The dotted line corresponds to the negative control.(PDF) ppat.1007315.s014.pdf (390K) GUID:?DA65919B-9C24-467A-B70F-68F9DE87118F S6 Fig: YFP-DHH1 localization after treatment with different benzoxaboroles. These are not “typical” images; instead, fields in which nuclear periphery granules were present have been chosen. Some (but not all) examples of peri-nuclear granule patterns are indicated by arrows. Compounds used are shown on each image. For AN7973 examples from the three different experiments are shown. The key is on the bottom left.(TIF) ppat.1007315.s015.tif (3.7M) GUID:?12DE6DD0-7E1F-4A15-A424-A083555C8651 S7 Fig: YFP-DHH1 localization after treatment with different benzoxaboroles. These are not “typical” images; instead, fields in which nuclear periphery granules were present have been chosen. Compounds used are indicated and the key is as in S6 Fig. The control image in this case had brighter YFP fluorescence, for unknown reasons.(TIF) ppat.1007315.s016.tif (4.1M) GUID:?249E9D68-EA4B-49F5-A4F8-FA415EB10C0B S8 Fig: Sequences of CPSF3 from (TREU927), and sequence is present because of a gap in the genome assembly.(PDF) ppat.1007315.s017.pdf (723K) GUID:?5A535817-3B10-4D72-AD9A-F1C75B568E33 S9 Fig: Domain annotations, residues linked to resistance mutations in and mapped onto a structural model of sequence. Grey dots indicate the predicted binding pocket for AN7973 for reference. C. Overlay of two snapshots from the first (slowest) mode obtained by normal mode analysis of the numbering) are shown as red sticks for reference. (PNG) ppat.1007315.s018.png (959K) GUID:?DA782AA4-98AD-47EE-98F6-37744A0AB4C7 S10 Fig: Details of EC50 results in cells with and without CPSF3-myc expression. (A) Induced expression of Myc-tagged RBSR4 and U2AF35.(B) IC50 measurements in cells with tetracycline-inducible expression of myc-CPFS3. (PDF) ppat.1007315.s019.pdf (753K) GUID:?7F51827F-57EF-43E2-9171-E8F2434E71A0 S11 Fig: Induced fit docking results for AN7973 in the active site of TbCPSF3. AN7973 (cyan sticks) was docked in the negatively charged form (A, tetrahedral geometry) Rabbit Polyclonal to CNNM2 and the neutral form (B, trigonal planar geometry). In both cases, the top scoring poses with Glide docking scores of -9.3 kcal/mol and -8.4 kcal/mol, respectively, are shown. For the tetrahedral geometry, in total, two poses with an average docking score of -9.00.4 kcal/mol and for the planar geometry, three poses with an average docking score of -6.91.4 kcal/mol were obtained. The TbCPSF3 homology model is shown in grey cartoon representation with important interacting residues highlighted as sticks and the zinc ions as grey spheres. Dashed lines indicate metal-coordination bonds (grey), metal-ligand interactions (black) and hydrogen bonds to the ligand (purple). The residue numbers correspond to the CPSF3 sequence.(PNG) ppat.1007315.s020.png (1.1M) GUID:?69B4382F-9B0F-4B6D-94CB-A464B54B7F5C S12 Fig: Local quality estimate by QMEAN score mapped to the protein structures of hCPSF73 (template) and the homology models of splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of with AN7973 inhibited splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear.Only in the loop 378C385 (or 403C410 in the numbering) are two valine residues replaced by isoleucine, which might slightly restrain the space accessible to the compound in the active site (S8 Fig). Our results suggested a mechanistic link between splicing inhibition and accumulation of specific methylated metabolites (Fig 7B). (XLSX) ppat.1007315.s008.xlsx (10K) GUID:?C76785C2-C31E-4A05-9550-0B9680309558 S9 Table: Global validation of homology models. Details are in the Table.(XLSX) ppat.1007315.s009.xlsx (10K) GUID:?F5677B43-FB27-49CA-A91A-F67DF96BA016 S1 Fig: Statistical analysis of results in Fig 3. VTP-27999 A: Results for an initial experiment in which the level of Y structure was quantified relative to in the presence and absence of benzoxaboroles. (PDF) ppat.1007315.s011.pdf (408K) GUID:?46DAE9FC-6E9C-4125-836D-413BA199EF44 S3 Fig: Time-to-kill assays for benzoxaboroleslike S1 Fig but with more compounds and without the untreated control. (PDF) ppat.1007315.s012.pdf (77K) GUID:?A0873AD0-75AE-403F-94FA-70C33B603A40 S4 Fig: Effects of benzoxaboroles on Y structure levels. A. Time course of effects of AN7973, AN3967, AN5827 and AN11736 in bloodstream forms (Experiment 2 in Fig 7). Sinefungin treatment was for 30 min.B, C: effect of AN7973 in procyclic forms. (PDF) ppat.1007315.s013.pdf (377K) GUID:?066C3D3A-7295-4810-9CAB-5FD36B72BE59 S5 Fig: Results for three separate experiments (A, B and C) in which the location of YFP-DHH1 was analysed. The average percentages of cells with clear (dark blue) or possible (pale blue) peri-nuclear granules are plotted as bars, with the average numbers of large granules (at least 4 contiguous pixels at maximum intensity) anywhere in the cell displayed as black spots. The dotted line corresponds to the negative control.(PDF) ppat.1007315.s014.pdf (390K) GUID:?DA65919B-9C24-467A-B70F-68F9DE87118F S6 Fig: YFP-DHH1 localization after treatment with different benzoxaboroles. These are not “typical” images; instead, fields in which nuclear periphery granules were present have been chosen. Some (but not all) examples of peri-nuclear granule patterns are indicated by arrows. Compounds used are demonstrated on each image. For AN7973 good examples from your three different experiments are demonstrated. The key is definitely on the bottom remaining.(TIF) ppat.1007315.s015.tif (3.7M) GUID:?12DE6DD0-7E1F-4A15-A424-A083555C8651 S7 Fig: YFP-DHH1 localization after treatment with different benzoxaboroles. These are not “standard” images; instead, fields in which nuclear periphery granules were present have been chosen. Compounds used are indicated and the key is as in S6 Fig. The control image in this case experienced brighter YFP fluorescence, for unfamiliar reasons.(TIF) ppat.1007315.s016.tif (4.1M) GUID:?249E9D68-EA4B-49F5-A4F8-FA415EB10C0B S8 Fig: Sequences of CPSF3 from (TREU927), and sequence is present because of a space in the genome assembly.(PDF) ppat.1007315.s017.pdf (723K) GUID:?5A535817-3B10-4D72-AD9A-F1C75B568E33 S9 Fig: Website annotations, residues linked to resistance mutations in and mapped onto a structural model of sequence. Grey dots show the expected binding pocket for AN7973 for research. C. Overlay of two snapshots from your first (slowest) mode obtained by normal mode analysis of the numbering) are demonstrated as reddish sticks for research. (PNG) ppat.1007315.s018.png (959K) GUID:?DA782AA4-98AD-47EE-98F6-37744A0AB4C7 S10 Fig: Details of EC50 results in cells with and without CPSF3-myc expression. (A) Induced manifestation of Myc-tagged RBSR4 and U2AF35.(B) IC50 measurements in cells with tetracycline-inducible expression of myc-CPFS3. (PDF) ppat.1007315.s019.pdf (753K) GUID:?7F51827F-57EF-43E2-9171-E8F2434E71A0 S11 Fig: Induced fit docking results for AN7973 in the active site of TbCPSF3. AN7973 (cyan sticks) was docked in the negatively charged form (A, tetrahedral geometry) and the neutral form (B, trigonal planar geometry). In both instances, the top rating poses with Glide docking scores of -9.3 kcal/mol and -8.4 kcal/mol, respectively, are demonstrated. For the tetrahedral geometry, in total, two poses with an average docking score of -9.00.4 kcal/mol and for the planar geometry, three poses with an average docking score of -6.91.4 kcal/mol were acquired. The TbCPSF3 homology model is definitely demonstrated in gray cartoon representation with important interacting residues VTP-27999 highlighted as sticks and the zinc ions as gray spheres. Dashed lines show metal-coordination bonds (gray), metal-ligand relationships (black) and hydrogen bonds to the ligand (purple). The residue figures correspond to the CPSF3 sequence.(PNG) ppat.1007315.s020.png (1.1M) GUID:?69B4382F-9B0F-4B6D-94CB-A464B54B7F5C S12 Fig: Local quality estimate by QMEAN score mapped to the protein structures of hCPSF73 (template) and the homology models of splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of with AN7973 inhibited splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and build up of peri-nuclear granules. Methylation of the spliced innovator precursor RNA was not affected, but more long term AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Collectively, the results indicate that mRNA processing is definitely a primary target of AN7973. Polyadenylation is required.