Supporting Information. Fast and Efficient Fragment-Based Lead Generation. by Fully Automated Processing and Analysis of

Transcription

1 Supporting Information Fast and Efficient Fragment-Based Lead Generation by Fully Automated Processing and Analysis of Ligand-Observed NMR Binding Data Chen Peng, Alexandra Frommlet, Manuel Perez, Carlos Cobas, Anke Blechschmidt, Santiago Dominguez, and Andreas Lingel Mestrelab Research, S.L Feliciano Barrera 9B Baixo, Santiago de Compostela, Spain; Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, CA 94608, USA The Supporting Information includes four figures: Figure S1: Illustration of common ways to analyze ligand-observed experiments Figure S2: Illustration of spectral alignment between reference and screening spectra Figure S3: Demonstration of the normalization process of spectral pairs Figure S4: Comparison of manual and automatic analysis of F screening data Page S1

2 Figure S1 a) 1 H-observed b) F-observed Ref3 Ref2 Ref1 T1ρ short Ref7 Ref6 Ref5 Ref4 T1ρ long STD p1 p2 p3 p4 p5 p6 p1 p2 p3 p4 Figure S1: Illustration of common ways to analyze ligand-observed experiments. Yellow peaks represent reference spectra, black peaks screening spectra and blue boxes indicate positive hits. (a) T1ρ and STD experiments are performed to observe changes in proton signals. As observed for p1 and p6, a peak (p) decrease between the T1ρ short and T1ρ long spectra recorded on the mixture sample in the presence of protein is interpreted as binding. In contrast, p2, p3, p4 and p5 are unchanged, indicating that the corresponding compounds are not binding. In the difference of the STD experiment, peaks with positive intensity indicate that the corresponding compound is binding. The reference 1 H spectra (Ref1-Ref3) of the individual ligands are stacked together with the mixture spectra to assign the individual peaks to ligands. As a result, Ref2 is classified as a hit (blue), and Ref1 and Ref3 are not hits. (b) A F CPMG experiment is run on a sample containing fluorinated compounds in the absence and presence of protein, and then individual peaks are assessed. Signals of p2 and p3 are decreased in the protein containing sample, whereas p1 and p4 are unchanged, indicating that compounds 5 and 7 correspond to positive hits. As described for panel a), the reference F spectra (Ref4-Ref7) of the individual ligands are stacked together with the mixture spectra to assign mixture peaks to ligands. Page S2

3 Figure S2 T1rho short T1rho long T1rho short T1rho long 1 H [ppm] Figure S2: Spectral alignment of reference and screening spectra. Top panel: 1 H reference (blue) and screening experiment spectra (red and yellow spectra representing a T1ρ pair of short and long spin-lock experiments) often display a systematic offset. To address this, a signal with a defined chemical shift value, e.g. DSS with a signal at 0 ppm, is added to the samples and serves as an internal reference signal. This allows the determination of the offset value. Bottom panel: The offset is then applied as a correction factor. Afterwards, the spectra are well aligned and peaks can be properly matched in the subsequent analysis. Page S3

4 Figure S3 1 H [ppm] Figure S3: Normalization of spectral pairs. A T1ρ experiment typically consists of a pair of spectra measured on the same sample recorded with a short and long spin-lock time (shown in brown and green, respectively). To normalize the intensity of the spectra, a reference signal which is present in both samples is integrated and normalized to a defined value. Usually, the DMSO signal (boxed in red) is utilized for this purpose. In order to avoid artefactual DMSO normalization in case ligand signals are in close proximity, the user should define the region for DMSO peak normalization as narrow as possible. In the top panel, the spectra are shown after spectral alignment (see main text and Figure S1) but before normalization. After normalization (lower panel), the intensities of the DMSO signal in the two spectra are of the same intensity. This step is critical to obtain correct integral values of compound peaks in the subsequent analysis. Page S4

5 Figure S4 a) Signal reduction of F signal in manual analysis b Hit /present No hit /present No hit /hit Hit /hit Signal reduction of F signal in automatic analysis b) c) c Used by manual analysis Reference Used by automatic analysis Ref2 Ref1 0.75(m) 0.00(a) N/A(m) 0.54(a) 0.8(m) 0.5(a) F [ppm] F [ppm] Figure S4: Comparison of manual and automatic analysis of F screening data. a) For each of the 551 compounds evaluated, the signal reduction determined by the manual analysis is plotted on the y-axis, whereas the value determined by the automatic analysis is plotted on the x-axis (normalized values, a value of 1 represents no signal reduction of the compound peak in the experiment with protein present compared to the experiment performed in the absence of protein, a value of 0 corresponds to complete loss of signal in the experiment containing protein). The solid diagonal line represent equal values (ratio of 1), and the dotted lines are drawn at 3 fold deviation from the equivalent value line. The hit criteria for both analyses was set to a reduction of more than 0.4 (40%), indicated by a grey line. Compounds which were evaluated with the same conclusion No Hit /present are colored grey (the reduction values for these compounds were not determined in the manual analysis), and compounds with the same conclusion Hit /hit are colored green. The majority of false positives ( No hit /hit, blue) and false negatives ( Hit /present, red) are within 10-20% of the threshold value, as described in the main text. Two outliers are labeled b and c and explained in more detail in panel S4b and S4c. b) Example of a case where the manual analysis concluded Hit, whereas the automatic analysis determined present ( false negative ). The reason for the discrepancy is a disconnect between the reference (yellow) and the mixture spectra (black) of this particular compound. The compound displays a major and minor peak in the reference, but only a single peak at the position of the minor peak in the mixture spectra. In the manual analysis, the peak change was determined on the signal visible in the mixture spectra and a reduction of 75% was determined. On the contrary, the automatic analysis picked only the major peak (the intensity of the minor peak was below the noise threshold and excluded) and therefore worked on a region of the corresponding to the major peak, where no significant changes are observed. c) Example of a case where the manual analysis concluded No hit, whereas the automatic analysis determined hit ( false positive ). Due to close proximity of the signals of two different compounds, the peak change determined by the automatic analysis is influenced by the neighboring signal. The conclusions are consistent for the compound that experiences the signal change (Ref2, right peak), but the signal change of the neighboring compound (Ref1, left peak) is influenced during integration in the automatic analysis, leading to inconsistent conclusions. Page S5