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

Page S1

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, 15706 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 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

corre-sponding compounds are not binding. In the difference spectrum 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 19_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 19_{F spectra (Ref4-Ref7) of the individual ligands are}

stacked together with the mixture spectra to assign mixture peaks to ligands.

Ref4 Ref5 Ref6 Ref7 p1 p2 p3 p4 Ref1 STD Ref2 Ref3

a)

b)

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. 1_{H [ppm]} ∆ ∆ Reference spectrum Reference spectrum

T1rhoshort spectrum

T1rholong spectrum

Reference spectrum

Reference spectrum

T1rhoshort spectrum

T1rholong spectrum

Figure S3: Normalization of spectral pairs. A T1ρ experiment typically consists of a pair of spectra meas-ured 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 sam-ples 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.

1_{H [ppm]}

Signal reduction of 19_{F signal in automatic analysis} Signal re duction of 19F signal in manual analysis 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

“No hit”/present

“No hit”/hit

“Hit”/present

“Hit”/hit

Figure S4: Comparison of manual and automatic analysis of 19_{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

analy-sis), 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

pres-ent (’false negative’). The reason for the discrepancy is a disconnect between the reference spectrum (yellow) and

the mixture spectra (black) of this particular compound. The compound displays a major and minor peak in the reference spectrum, 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 spectrum 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 b) c) 19_{F [ppm]} 19_{F [ppm]} -62.6 -62.8 -63.0 -64.10 -64.15 -64.20 CPMGblank CPMGprotein CPMGblank CPMGprotein b c Reference Ref2 Ref1

Used by automatic analysis Used by manual analysis

0.75(m) 0.00(a)

N/A(m)