Journal: bioRxiv

Article Title: AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

doi: 10.1101/2023.06.14.544560

Figure Lengend Snippet: ( A ) Schematic overview of the LuTHy-BRET and LuTHy-LuC assays. X: Protein X, Y: Protein Y, D: NanoLuc donor, A: mCitrine acceptor, AB: antibody. ( B ) With the LuTHy assay, each protein pair X-Y can be tested in eight possible configurations (N- vs. C-terminal fusion for each protein), and proteins can be swapped from one tag to the other resulting in 16 quantitative scores for each protein pair, i.e. eight for LuTHy-BRET and eight for LuTHy-LuC. ( C ) Line plots showing the fraction of protein pairs that scored positive (y-axis) dependent on the quantitative interaction scores (x-axis) for 10 binary PPI assay versions. For each tested protein pair, the tagging configuration with the highest interaction score is used. For LuTHy all eight tagging configurations were tested, whereas for MN2H, VN2H, YN2H, GPCA, NanoBi four and for KISS, MAPPIT and SIMPL two tagging configurations were tested. Recovery rates at maximum specificity, i.e. where none of the protein pairs in the RRS scored positive (0%), are indicated. Note that in Choi et al. recovery rates at maximum specificity were calculated by using distinct cut-offs for each tagging configuration. ( D ) Line plots showing the fraction of protein pairs that scored positive (y-axis) dependent on the distribution of interaction scores, i.e. the mean of all interaction scores + n*(sd) (x-axis) for 10 binary PPI assay versions. Recovery rates at mean + 1 standard deviation are indicated ( E ) Line plots showing the fraction of protein pairs that scored positive (y-axis) dependent on the distribution of interaction scores, i.e. the median of all interaction scores + n*(sd) (x-axis) for 10 binary PPI assays. Recovery rates at median + 1 standard deviation are indicated. LuTHy data from this study; SIMPL from Yao et al ; all other from Choi et al . Note that the SIMPL assay was benchmarked by Yao et al against 88 positive proteins pairs derived from the hsPRS-v1 and as a random reference set against “88 protein pairs with baits and preys selected from the PRS but used in combinations determined computationally to have low probability of interaction” .

Article Snippet: For the mN2H assay, additional control plasmids (pDEST-N2H-F1-empty vector, Addgene #125551; pDEST-N2H-F2-empty vector, Addgene #125552) were used, as previously described ( ).

Techniques: Standard Deviation, Derivative Assay

Journal: bioRxiv

Article Title: AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

doi: 10.1101/2023.06.14.544560

Figure Lengend Snippet: ( A ) Scatter plot showing PAE (x-axis) against interface area (y-axis) for all hsPRS-AF (blue) and hsRRS-AF (magenta) protein pairs. Average classifier probability from the 50 maSVM models is displayed as the size of the data points and as a colored grid in the background. ( B ) Scatter plots showing PAE (x-axis, left panel) or interface area (x-axis, right panel) against classifier probability (y-axis) for all hsPRS-AF (blue) and hsRRS-AF (magenta) protein pairs. ( C ) Bar plots showing the fraction of hsPRS-AF and hsRRS-AF protein pairs that scored above classifier probabilities of 50%, 75% and 95%. ( D ) Bar plots showing the fraction of hsPRS-AF and hsRRS-AF interactions with structures deposited in PDB that scored above classifier probabilities of 50%, 75% and 95% by AlphaFold-Multimer (i) and the fraction of hsPRS-v2 and hsRRS-v2 interactions with structures deposited in PDB that scored above classifier probabilities of 50%, 75% or 95% by LuTHy (ii) or the union of five other binary assays (iii), N2H (MN2H, VN2H, YN2H), GPCA, KISS, MAPPIT and NanoBiT. Data for the SIMPL assay was excluded for this analysis due to the different composition of the reference sets.

Article Snippet: For the mN2H assay, additional control plasmids (pDEST-N2H-F1-empty vector, Addgene #125551; pDEST-N2H-F2-empty vector, Addgene #125552) were used, as previously described ( ).

Techniques:

Journal: bioRxiv

Article Title: AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

doi: 10.1101/2023.06.14.544560

Figure Lengend Snippet: ( A-H ) Boxplots of assay specific interaction scores before and after robust scaler normalization for the multiprotein complex proteins in all tagging configurations of donor and acceptor constructs. Boxplots display the constructs’ median, lower and upper hinges the 25 th and 75 th percentiles, lower and upper whiskers extending from the hinges with 1.5x the inter-quartile range and outlier points beyond the end of the whiskers. The thick horizontal line indicates the median interaction score over all constructs of the multiprotein complex set and the dashed lines the respective IQR of the 25 th and 75 th quartiles. Note that the horizontal lines always refer to the median and IQR before normalization and that the range of the y-axis is limited to visualize all boxplots as well as the median and IQR, but high scoring protein pairs (outliers) are hidden. ( A ) cBRET ratios for donor constructs before (top) and after (bottom) robust scaler normalization. ( B ) cBRET ratios for acceptor constructs before (top) and after (bottom) robust scaler normalization. ( C ) cLuC ratios for donor constructs before (top) and after (bottom) robust scaler normalization. ( D ) cLuC ratios for acceptor constructs before (top) and after (bottom) robust scaler normalization. ( E ) Luminescence after co-precipitation (NL OUT ) for donor constructs before (top) and after (bottom) robust scaler normalization. ( F ) Luminescence after co-precipitation (NL OUT ) for acceptor constructs before (top) and after (bottom) robust scaler normalization. ( G ) mN2H ratios for F1 constructs before (top) and after (bottom) robust scaler normalization. ( H ) mN2H ratios for F2 constructs before (top) and after (bottom) robust scaler normalization.

Article Snippet: For the mN2H assay, additional control plasmids (pDEST-N2H-F1-empty vector, Addgene #125551; pDEST-N2H-F2-empty vector, Addgene #125552) were used, as previously described ( ).

Techniques: Construct

Journal: bioRxiv

Article Title: AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

doi: 10.1101/2023.06.14.544560

Figure Lengend Snippet: ( A ) Structures of the protein complexes analyzed in this study: LAMTOR (PDB: 6EHR), MIS12 (PDB: 5LSK), and BRISC (PDB: 6H3C). ( B ) Binary interaction approach to systematically map PPIs within distinct complexes. Every protein subunit from each complex was screened against every other one (all-by-all, 16×16 matrix). ( C-E ) Scatter plot showing ( C ) in-cell mCitrine expression (x-axis) against cBRET ratios (y-axis), ( D ) luminescence after co-precipitation (NL OUT ) (x-axis) against cLuC ratios (y-axis) or ( E ) the number of protein pairs (x-axis) against the mN2H ratios (y-axis) for all intra-complex (blue) and inter-complex (magenta) protein pairs from all eight tagging configurations. Average classifier probabilities from the 50 maSVM models are displayed as the size of the data points and as a colored grid in the background. ( F-H ) Scatter plot showing on the x-axis the ( F ) cBRET ratios, ( G ) cLuC ratios or ( H ) mN2H ratios against classifier probabilities (y-axis) for all intra-complex (blue) and inter-complex (magenta) protein pairs from all eight tagging configurations. ( I-K ) Bar plots showing the fraction of intra-complex and inter-complex protein pairs that scored above the classifier probabilities of 50%, 75% or 95% by ( I ) LuTHy-BRET, ( J ) LuTHy-LuC and ( K ) mN2H. Only the highest classifier probability per tested tagging configuration is considered. ( L ) Heatmaps showing the classifier probabilities for the Donor/F1 protein pairs (x-axis) against the Acceptor/F2 protein pairs (y-axis) for LuTHy-BRET (orange, left), LuTHy-LuC (purple, middle) and mN2H (green, right). Only the highest classifier probability per tested tagging configuration is shown. Not expressed constructs are filled black and protein names colored in red.

Article Snippet: For the mN2H assay, additional control plasmids (pDEST-N2H-F1-empty vector, Addgene #125551; pDEST-N2H-F2-empty vector, Addgene #125552) were used, as previously described ( ).

Techniques: Expressing, Construct