db3  (Biolog Inc)

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a , To design new ligand-induced protein interactions, potential interface sites are first identified on the target protein–ligand complex. The corresponding patches are then used to find complementary fingerprints in a patch database. The top patches are aligned and scored to refine the selection. Associated binding motifs (seeds) undergo sequence optimization with an emphasis on designing new hydrogen bond networks with the target protein and small molecule. Seeds are then grafted on suitable scaffolds from a structural database, and the rest of the scaffold interface is redesigned using Rosetta. Finally, the top (approximately) 2,000 designs, according to different structural metrics, are selected and screened experimentally. b , Target candidates in complex with their respective small molecules (top row). Neosurfaces with their protein binding propensities (bottom row). Sites selected for binder design are highlighted with dashed circles. c , Diversity of the computational designs mapped using multidimensional scaling (MDS) of pairwise r.m.s.d. values between all designs. Experimentally confirmed binders are highlighted with a star. In total, 1,995 computational designs were plotted for Bcl2–venetoclax (Bcl2–Ven), 1,998 for DB3–progesterone (DB3–Pro) and 1,997 for PDF1–actinonin (PDF1–Act).

Article Snippet: The ZF438-DB3 scFv (V H /V L ) fusion protein was expressed using a PURExpress kit from NEB (E6800S) with the addition of a disulfide bond enhancer (E6820S).

Techniques: Selection, Binding Assay, Sequencing, Protein Binding

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a , Models of the designed binders in complex with their respective target complexes: Bcl2–venetoclax, DB3–progesterone and PDF1–actinonin. b , Histograms of the binding signal (phycoerythrin; PE) measured by flow cytometry on yeast displaying the designed binders. Yeast were either unlabelled or labelled with 500 nM of the respective target protein preincubated with the ligand or with the target protein alone. c , Histograms of the binding signal (PE) measured by flow cytometry on yeast displaying designed binders, a mutated version with a single-point mutant at the predicted interface and the starting scaffold used for the design process. Yeast cells were labelled with 500 nM of their respective ligand–protein complex. Dashed lines represent the geometric mean of the designed binder signal. d , Binding measured on yeast displaying DBVen1619_1, DBPro1156_1 or DBAct553_1 labelled with the target protein alone (grey), the target protein in complex with the original small molecule (blue) or the target protein in complex with the small-molecule analogue (magenta). Control analogues were S55746, OBz-Pro and TBDMS-Act. Detailed structures of the small molecules and their analogues are shown in Supplementary Fig. .

Article Snippet: The ZF438-DB3 scFv (V H /V L ) fusion protein was expressed using a PURExpress kit from NEB (E6800S) with the addition of a disulfide bond enhancer (E6820S).

Techniques: Binding Assay, Flow Cytometry, Mutagenesis, Control, Analogues

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a , Computational model coloured with the average enrichment score in the SSM for each amino acid position of the designed binder. Red indicates that an amino acid position is sensitive to mutations, whereas blue indicates a more tolerant amino acid position. Target proteins are shown in grey. b , Affinity measurements for DBVen1619_2, DBPro1156_2 and DBAct553_2 by biolayer interferometry. Each measurement was obtained in the presence (orange) or absence (blue) of the respective small molecule. The fits were calculated using a nonlinear four-parameter curve-fitting analysis. c , Crystal structure of DBAct553_1 in complex with actinonin-bound PDF1 (PDB 8S1X ). The computational model (light pink) is aligned with the crystal structure (magenta). Inset shows the alignment of the residues at the interface. d , Cryo-electron microscopy (cryo-EM) structure obtained for DBPro1156_2 in complex with progesterone (prog.)-bound DB3.The computational model (light blue) is aligned with the cryo-EM structure (dark blue). Inset shows the alignment of the residues at the interface.

Article Snippet: The ZF438-DB3 scFv (V H /V L ) fusion protein was expressed using a PURExpress kit from NEB (E6800S) with the addition of a disulfide bond enhancer (E6820S).

Techniques: Cryo-Electron Microscopy, Cryo-EM Sample Prep

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a , Schematic of the cell-free expression system with scFv DB3 fused to a zinc-finger transcription factor and DBPro1156_2 fused to T7 RNA polymerase (Pol). b , Fluorescence (relative fluorescence units; RFU) measured with each monomeric component or mixed, with or without 20 μM progesterone. c , Progesterone-dose-dependent responses performed in a cell-free system containing both components. d , Schematic of the split NanoLuc system functionalizing DBAct553_1 and PDF1. e , Intracellular NanoLuc luminescence of HEK293T transfected with C-terminal split NanoLuc-fused PDF1 only, N-terminal split NanoLuc-fused DBAct553_1 only or both together, with or without 10 μM actinonin. f , Actinonin-dose-dependent responses performed on HEK293T transfected with both components. g , Schematic representation of αHER2-specific 2G-CAR and the drug-inducible αHER2-CAR split system (split CID-CAR). The two domains assembled upon addition of venetoclax. h , Killing efficiency of CAR-T cells with and without venetoclax using untransduced (UT) murine primary T cells or cells transduced with 2G-CAR or the split CID-CAR. Tumour cell lysis was measured after 48 h of coincubation with target cells. The percentage of live target cells was normalized to the number of live cells in each well at t = 0 h and further normalized to the growth of target cells cultured alone. i , Killing efficiency of CAR-T cells measured over time. Tumour cell counts at different time points were normalized to the number of live cells in each well at t = 0 h. A concentration of 10 nM venetoclax was used. Two-way ANOVA with Tukey’s multiple comparison test; NS, not significant. **** P < 0.0001. Data are presented as mean ± s.d. Data points are derived from three technical replicates ( a – f ) or three biological replicates ( g – i ).

Article Snippet: The ZF438-DB3 scFv (V H /V L ) fusion protein was expressed using a PURExpress kit from NEB (E6800S) with the addition of a disulfide bond enhancer (E6820S).

Techniques: Expressing, Fluorescence, Transfection, Transduction, Lysis, Cell Culture, Concentration Assay, Comparison, Derivative Assay

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a . Schematic of the cell free-expression system with PDF1 protein fused to a zinc finger transcription factor and DBAct553_1 fused to T7 RNA polymerase. b . Fluorescence (Relative fluorescence unit; RFU) measured in wells containing each monomeric component or mixed, without or with 16 μM Actinonin. p < 0.0001 (****). c . Actinonin dose-dependent responses performed in a cell free system containing both components. d . Schematic of the extracellular split NanoLuc system functionalizing DB3 scFv and DBPro1156_2. e . Extracellular NanoLuc luminescence of HEK293T transfected with C-term split NanoLuc-fused DB3, N-term split NanoLuc-fused DBPro1156_2 or both together without or with 25 μM Progesterone. f . Progesterone dose-dependent responses performed on HEK293T transfected with split-NanoLuc DB3 scFv and DBPro1156_2. p < 0.0001 (****). g . Schematic of the intracellular split NanoLuc system functionalizing Bcl2 and DBVen1619_2. h . Intracellular NanoLuc luminescence of HEK293T transfected with C-term split NanoLuc-fused Bcl2 only, N-term split NanoLuc-fused DBVen1619_2 only or both together without or with 1 μM Venetoclax. i . Venetoclax dose-dependent responses performed on HEK293T transfected with split-NanoLuc Bcl2 and DBVen1619_2. j . Schematic of the GEMS reporter system functionalizing Bcl2-based CID. Both protein components of the CID are individually fused to erythropoietin receptor (EpoR) chains linked to an intracellular human IL6RB domain, which induces the expression of a reporter gene (secreted NanoLuc luciferase) when activated. k . NanoLuc luminescence of HEK293T cells transfected with Bcl2-GEMS only, DBVen1619_2 only or both together without or with 12 nM Venetoclax. p < 0.0001 (****). l . Venetoclax dose-dependent responses performed on HEK293T transfected with Bcl2 and DBV1619 GEMS receptors. p < 0.0001 (****). Two-way ANOVA with Tukey’s multiple comparison test, non-significant (ns). Barplots are presented as mean ± standard deviations. Data points are derived from three technical replicates (a-i) or three biological replicates (j-l).

Article Snippet: The ZF438-DB3 scFv (V H /V L ) fusion protein was expressed using a PURExpress kit from NEB (E6800S) with the addition of a disulfide bond enhancer (E6820S).

Techniques: Expressing, Fluorescence, Transfection, Luciferase, Comparison, Derivative Assay

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a , To design new ligand-induced protein interactions, potential interface sites are first identified on the target protein–ligand complex. The corresponding patches are then used to find complementary fingerprints in a patch database. The top patches are aligned and scored to refine the selection. Associated binding motifs (seeds) undergo sequence optimization with an emphasis on designing new hydrogen bond networks with the target protein and small molecule. Seeds are then grafted on suitable scaffolds from a structural database, and the rest of the scaffold interface is redesigned using Rosetta. Finally, the top (approximately) 2,000 designs, according to different structural metrics, are selected and screened experimentally. b , Target candidates in complex with their respective small molecules (top row). Neosurfaces with their protein binding propensities (bottom row). Sites selected for binder design are highlighted with dashed circles. c , Diversity of the computational designs mapped using multidimensional scaling (MDS) of pairwise r.m.s.d. values between all designs. Experimentally confirmed binders are highlighted with a star. In total, 1,995 computational designs were plotted for Bcl2–venetoclax (Bcl2–Ven), 1,998 for DB3–progesterone (DB3–Pro) and 1,997 for PDF1–actinonin (PDF1–Act).

Article Snippet: A chimeric DB3 Fab (Supplementary Table ) was produced using the Expi293 expression system from Thermo Fisher Scientific (A14635).

Techniques: Selection, Binding Assay, Sequencing, Protein Binding

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a , Models of the designed binders in complex with their respective target complexes: Bcl2–venetoclax, DB3–progesterone and PDF1–actinonin. b , Histograms of the binding signal (phycoerythrin; PE) measured by flow cytometry on yeast displaying the designed binders. Yeast were either unlabelled or labelled with 500 nM of the respective target protein preincubated with the ligand or with the target protein alone. c , Histograms of the binding signal (PE) measured by flow cytometry on yeast displaying designed binders, a mutated version with a single-point mutant at the predicted interface and the starting scaffold used for the design process. Yeast cells were labelled with 500 nM of their respective ligand–protein complex. Dashed lines represent the geometric mean of the designed binder signal. d , Binding measured on yeast displaying DBVen1619_1, DBPro1156_1 or DBAct553_1 labelled with the target protein alone (grey), the target protein in complex with the original small molecule (blue) or the target protein in complex with the small-molecule analogue (magenta). Control analogues were S55746, OBz-Pro and TBDMS-Act. Detailed structures of the small molecules and their analogues are shown in Supplementary Fig. .

Article Snippet: A chimeric DB3 Fab (Supplementary Table ) was produced using the Expi293 expression system from Thermo Fisher Scientific (A14635).

Techniques: Binding Assay, Flow Cytometry, Mutagenesis, Control, Analogues

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a , Computational model coloured with the average enrichment score in the SSM for each amino acid position of the designed binder. Red indicates that an amino acid position is sensitive to mutations, whereas blue indicates a more tolerant amino acid position. Target proteins are shown in grey. b , Affinity measurements for DBVen1619_2, DBPro1156_2 and DBAct553_2 by biolayer interferometry. Each measurement was obtained in the presence (orange) or absence (blue) of the respective small molecule. The fits were calculated using a nonlinear four-parameter curve-fitting analysis. c , Crystal structure of DBAct553_1 in complex with actinonin-bound PDF1 (PDB 8S1X ). The computational model (light pink) is aligned with the crystal structure (magenta). Inset shows the alignment of the residues at the interface. d , Cryo-electron microscopy (cryo-EM) structure obtained for DBPro1156_2 in complex with progesterone (prog.)-bound DB3.The computational model (light blue) is aligned with the cryo-EM structure (dark blue). Inset shows the alignment of the residues at the interface.

Article Snippet: A chimeric DB3 Fab (Supplementary Table ) was produced using the Expi293 expression system from Thermo Fisher Scientific (A14635).

Techniques: Cryo-Electron Microscopy, Cryo-EM Sample Prep

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a , Schematic of the cell-free expression system with scFv DB3 fused to a zinc-finger transcription factor and DBPro1156_2 fused to T7 RNA polymerase (Pol). b , Fluorescence (relative fluorescence units; RFU) measured with each monomeric component or mixed, with or without 20 μM progesterone. c , Progesterone-dose-dependent responses performed in a cell-free system containing both components. d , Schematic of the split NanoLuc system functionalizing DBAct553_1 and PDF1. e , Intracellular NanoLuc luminescence of HEK293T transfected with C-terminal split NanoLuc-fused PDF1 only, N-terminal split NanoLuc-fused DBAct553_1 only or both together, with or without 10 μM actinonin. f , Actinonin-dose-dependent responses performed on HEK293T transfected with both components. g , Schematic representation of αHER2-specific 2G-CAR and the drug-inducible αHER2-CAR split system (split CID-CAR). The two domains assembled upon addition of venetoclax. h , Killing efficiency of CAR-T cells with and without venetoclax using untransduced (UT) murine primary T cells or cells transduced with 2G-CAR or the split CID-CAR. Tumour cell lysis was measured after 48 h of coincubation with target cells. The percentage of live target cells was normalized to the number of live cells in each well at t = 0 h and further normalized to the growth of target cells cultured alone. i , Killing efficiency of CAR-T cells measured over time. Tumour cell counts at different time points were normalized to the number of live cells in each well at t = 0 h. A concentration of 10 nM venetoclax was used. Two-way ANOVA with Tukey’s multiple comparison test; NS, not significant. **** P < 0.0001. Data are presented as mean ± s.d. Data points are derived from three technical replicates ( a – f ) or three biological replicates ( g – i ).

Article Snippet: A chimeric DB3 Fab (Supplementary Table ) was produced using the Expi293 expression system from Thermo Fisher Scientific (A14635).

Techniques: Expressing, Fluorescence, Transfection, Transduction, Lysis, Cell Culture, Concentration Assay, Comparison, Derivative Assay

Journal: Nature

Article Title: Targeting protein–ligand neosurfaces with a generalizable deep learning tool

doi: 10.1038/s41586-024-08435-4

Figure Lengend Snippet: a . Schematic of the cell free-expression system with PDF1 protein fused to a zinc finger transcription factor and DBAct553_1 fused to T7 RNA polymerase. b . Fluorescence (Relative fluorescence unit; RFU) measured in wells containing each monomeric component or mixed, without or with 16 μM Actinonin. p < 0.0001 (****). c . Actinonin dose-dependent responses performed in a cell free system containing both components. d . Schematic of the extracellular split NanoLuc system functionalizing DB3 scFv and DBPro1156_2. e . Extracellular NanoLuc luminescence of HEK293T transfected with C-term split NanoLuc-fused DB3, N-term split NanoLuc-fused DBPro1156_2 or both together without or with 25 μM Progesterone. f . Progesterone dose-dependent responses performed on HEK293T transfected with split-NanoLuc DB3 scFv and DBPro1156_2. p < 0.0001 (****). g . Schematic of the intracellular split NanoLuc system functionalizing Bcl2 and DBVen1619_2. h . Intracellular NanoLuc luminescence of HEK293T transfected with C-term split NanoLuc-fused Bcl2 only, N-term split NanoLuc-fused DBVen1619_2 only or both together without or with 1 μM Venetoclax. i . Venetoclax dose-dependent responses performed on HEK293T transfected with split-NanoLuc Bcl2 and DBVen1619_2. j . Schematic of the GEMS reporter system functionalizing Bcl2-based CID. Both protein components of the CID are individually fused to erythropoietin receptor (EpoR) chains linked to an intracellular human IL6RB domain, which induces the expression of a reporter gene (secreted NanoLuc luciferase) when activated. k . NanoLuc luminescence of HEK293T cells transfected with Bcl2-GEMS only, DBVen1619_2 only or both together without or with 12 nM Venetoclax. p < 0.0001 (****). l . Venetoclax dose-dependent responses performed on HEK293T transfected with Bcl2 and DBV1619 GEMS receptors. p < 0.0001 (****). Two-way ANOVA with Tukey’s multiple comparison test, non-significant (ns). Barplots are presented as mean ± standard deviations. Data points are derived from three technical replicates (a-i) or three biological replicates (j-l).

Article Snippet: A chimeric DB3 Fab (Supplementary Table ) was produced using the Expi293 expression system from Thermo Fisher Scientific (A14635).

Techniques: Expressing, Fluorescence, Transfection, Luciferase, Comparison, Derivative Assay