Family wide chemical profiling and structural ... - media.nature.com · cDipartimento di Chimica e Tecnologia del Farmaco, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy;

Supplementary Notes, Figures and Tables accompanying

Family wide chemical profiling and structural

analysis of PARP and Tankyrase inhibitors

Elisabet Wahlberga,e

, Tobias Karlberga, Ekaterina Kouznetsova

a,f, Natalia Markova

a,g, Antonio

Macchiaruloc, Ann-Gerd Thorsell

a, Ewa Pol

b, Åsa Frostell

b, Torun Ekblad

a, Delal Öncü

d, Björn Kull

d,

Graeme Michael Robertsonc, Roberto Pellicciari

c, Herwig Schüler

a,1, and Johan Weigelt

a

aStructural Genomics Consortium, Karolinska Institutet, Department of Medical Biochemistry and

Biophysics, 17177 Stockholm, Sweden; bGE Healthcare Bio-Sciences AB, 75184 Uppsala, Sweden;

cDipartimento di Chimica e Tecnologia del Farmaco, University of Perugia, Via del Liceo 1, 06123 Perugia,

Italy; dActar AB, 17177 Stockholm, Sweden;

ePresent address: Faculty of Natural Resources and

Agricultural Sciences, Box 7082, 750 07 Uppsala, Sweden; fPresent address: University of Toronto, 101

College St, Toronto ON M5G 1L7,Canada; gPresent address: GE Healthcare Bio-Sciences AB, 75184

Uppsala, Sweden

These authors contributed equally to the manuscript: EW, TK, EK.

1Correspondence should be addressed to H.S. ([email protected]).

Nature Biotechnology: doi:10.1038/nbt.2121

2

Table of Contents

Supplementary Table 1 3 Supplementary Note 1: Comparison DSF vs. SPR for PARP15 and TNKS1 4

Supplementary Figure 1 4 Materials and Methods 5

Supplementary Table 2 6 Supplementary Note 2: Cross-validation of screening data with enzymatic assays and SPR 7

Supplementary Figure 2 7 Supplementary Table 4 7 Supplementary Figure 3 7 Supplementary Figure 4 8 Supplementary Table 5 9 Supplementary Table 6 9 Supplementary Figure 5 10 Supplementary Figure 6 10 Supplementary Table 7 11 Supplementary Figure 7 12 Summary 12 Materials and Methods 13

Supplementary Note 3: Principal Component Analysis and Multidimensional Scaling Study 14

Supplementary Table 8 14 Supplementary Table 9 14 Supplementary Table 10 15 Supplementary Figure 8 15 Supplementary Figure 9 16 Supplementary Table 11 17

Supplementary Note 4: Structural studies of TNKS2 and PARP14 using X-ray crystallography 18

Supplementary Figure 10 20 Supplementary Table 12 21 Supplementary Table 13 22 Materials and Methods 23

References 25


3

Supplementary Table 1. PARP domain crystal structures available in the Protein Data Bank.

PDB

code

Family

member Species Ligand Reference

1UK0# PARP1 H. sapiens FR257517 1

1UK1# PARP1 H. sapiens FR143829 2

1WOK# PARP1 H. sapiens compound10 in ref. 3 3

2RCW# PARP1 H. sapiens A620223 -

2RD6# PARP1 H. sapiens ABT-888 -

3GJW# PARP1 H. sapiens A968427 4

3GN7# PARP1 H. sapiens A861696 -

3L3L# PARP1 H. sapiens A906894 5

3L3M# PARP1 H. sapiens A927929 6

1A26 PARP1 G. gallus carba-NAD 7

1EFY PARP1 G. gallus compound 44 in ref. 8 8

2PAW PARP1 G. gallus - 9

1PAX PARP1 G. gallus DHQ 10

2PAX PARP1 G. gallus 4AN 9

3PAX PARP1 G. gallus 3-methoxybenzamide 9

4PAX PARP1 G. gallus NU1025 9

3KCZ# PARP2 H. sapiens 3-aminobenzamide 11

3KJD# PARP2 H. sapiens ABT-888 11

1GS0 PARP2 M. musculus - 12

3FHB# PARP3 H. sapiens 3-aminobenzoic acid 13

3C49# PARP3 H. sapiens KU0058948 13

3C4H# PARP3 H. sapiens DR2313 13

3CE0# PARP3 H. sapiens PJ34 13

3HKV PARP10 H. sapiens 3-aminobenzamide -

2PQF PARP12 H. sapiens 3-aminobenzoic acid -

2X5Y PARP13 H. sapiens - -

3GOY PARP14 H. sapiens 3-aminobenzamide This study

3SE2 PARP14 H. sapiens 6(5H)-phenanthridinone This study

3SMI PARP14 H. sapiens cpd 98 in this study This study

3SMJ PARP14 H. sapiens cpd 145 in this study This study

3GEY PARP15 H. sapiens PJ34 -

2RF5 TNKS1 H. sapiens - 14

3KR7 TNKS2 H. sapiens - 15

3KR8# TNKS2 H. sapiens XAV939 15

3MHJ# TNKS2 H. sapiens cpd 106 in this study This study

3MHK# TNKS2 H. sapiens cpd 183 in this study This study

3P0N# TNKS2 H. sapiens ACT20400 This study

3P0P# TNKS2 H. sapiens ACT8395 This study

3P0Q# TNKS2 H. sapiens ACT24188 This study

3U9H TNKS2 H. sapiens nicotinamide -

#Coordinates from these entries were used for generating Figure 2D of the main text (rendering of the

binding pocket space occupied by various PARP inhibitors).


4

Comparison Differential Scanning Fluorimetry (DSF) and Surface Plasmon Resonance

(SPR) assay to study inhibitor binding to PARP/ARTD enzymes

184 compounds were tested for interaction with the catalytic domains of PARP15 and TNKS1 using

Differential Scanning Fluorimetry (DSF)16,17

and Surface Plasmon Resonance (SPR).18

35 compounds were identified from the primary SPR analysis as binders to either protein. These were

subsequently subjected to a more detailed SPR analysis to determine binding kinetics. From the set of 35

compounds, it was possible to estimate binding kinetics for 15 compounds binding to both proteins, 15

compounds that bound only to TNKS1 and three compounds that bound only to PARP15. Two compounds

failed to yield reliable kinetics for either protein.

Supplementary Figure 1 shows the correlation between the thermal stabilizations measured by DSF and the

affinity measured by SPR. The overall correlation is good, however a significant fraction of the compounds

that yielded measurable binding constants by SPR failed to yield significant thermal shifts in the DSF assay.

SPR thus seems to be the more sensitive method and compounds yielding small, or negative, thermal shifts

may still bind the protein. Importantly, all compounds showing significant positive thermal shifts were

confirmed as binders by the SPR method.

Supplementary Figure 1. Correlation between thermal stabilization (Tm) and dissociation equilibrium constants

(pKD = - logKD) measured by SPR for compounds binding to PARP15 (green triangles) and TNKS1 (pink circles).

Error bars represent standard deviations (DSF data) and the standard error of fit for the SPR data (<15 % for all

data points and not clearly visible in the plot due to the logarithmic scale).


5

Materials and Methods

Protein expression and purification. Catalytic domain fragments of human TNKS1 and PARP15 proteins

were produced in Escherichia coli as hexahistidine fusions and purified using nickel affinity followed by

size exclusion chromatography. Proteins were typically >90% pure as judged by SDS-PAGE analysis.

Purified proteins were verified by Time-of-Flight Mass Spectrometry analysis.

Differential Scanning Fluorimetry. Proteins were diluted to 0.2 mg/ml in PBS buffer (pH 7.5) containing

2 mM TCEP and 1:1000 SyproOrange (Invitrogen). 25 l protein solution was added to all wells in 96-well

clear plates (BioRad), each well containing 0.5 µl pre-dispensed compound solution (2.5 mM dissolved in

DMSO). Optical Tape (Bio-Rad) was used to seal the plates. Thermal stability was measured by monitoring

SyproOrange fluorescence (excitation = 490 nm and excitation = 575 nm) while heating the samples from 20 to

90°C in increments of 1°C/min in a Bio-Rad iCycler, and the thermal shifts were determined as described17

using pure DMSO (2% v/v) as a reference. Each experiment was carried out in triplicate.

Surface plasmon resonance measurements. Experiments were carried out on a Biacore™ T200

instrument (GE Healthcare) at 25°C. Hexahistidine tagged TNKS2 or PARP15 was immobilized on a

Sensor Chip NTA (GE Healthcare) by first activating the chip with a 1 minute injection of 0.5 mM NiCl2 in

20 mM PBS, 0.05% surfactant P20, pH 7.4, followed directly by amine coupling of the hexahistidine tagged

protein (5 μg/ml in 20 mM PBS, 0.05% surfactant P20, pH 7.4) to a level of approximately 5000 resonance

units (RU). Binding of compounds was assessed by injecting 30 µM compound solution in running buffer

(20 mM PBS, 4 % DMSO, 0.05% surfactant P20, pH 7.4) over a reference surface, without NiCl2-activation

and without protein, and the immobilized surface. Injections were performed during 60 s, followed by a

dissociation period of 10 min. Dissociation equilibrium constants for the 35 identified binders were

determined by repeating the experiment using a standardized compound concentration series (0 to 200 μM)

and fitting of the data to a 1:1 binding model using Biacore evaluation software (GE Healthcare).


6

Supplementary Table 2. Recombinant protein constructs and assay buffer conditions for different proteins.

Protein Construct Assay buffer Comment

TNKS1 Q1091-Q1325 10 mM PBS, 2 mM TCEP, pH 7.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

TNKS2 G952-G1166 10 mM PBS, 2 mM TCEP, pH 7.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

PARP1 K654-L1013 20 mM HEPES, 300 mM NaCl, 2 mM TCEP pH 7.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

PARP2 D186-F530 20 mM Hepes, 300 mM NaCl, 10% Glycerol, 2 mM TCEP, pH 7.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

PARP3 K178-H532 20 mM Hepes, 300 mM NaCl, 10% Glycerol, 2 mM TCEP, pH 7.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

PARP4 A241-L600 20 mM HEPES, 2 mM TCEP pH 7.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

PARP9 K597-G799 20 mM Hepes, 300 mM NaCl, 10% Glycerol, 2 mM TCEP, pH 7.5 C-terminal His-tag, AHHHHHH

PARP10 A809-G1017 10 mM NaAc, 2 mM TCEP, pH 5.5 C-terminal His-tag, AHHHHHH

PARP12 G480-S688 10 mM PBS, 2 mM TCEP, pH 7.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

PARP13 K726-D896 20 mM HEPES, 300 mM NaCl, 2 mM TCEP pH 7.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

PARP14 H55-K248 10 mM NaAc, 2 mM TCEP, pH 5.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

PARP15 N247-A444 10 mM PBS, 2 mM TCEP, pH 7.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM

PARP16 M1-A279 10 mM NaAc, 2 mM TCEP, pH 5.5 N-terminal His-tag, MHHHHHHSSGVDLGTENLYFQSM


7

Cross-validation of screening data with enzymatic assays and SPR

I. Comparison Differential Scanning Fluorimetry (DSF) and enzymatic inhibition of PARP/ARTD enzymes

Five different compounds were tested for enzymatic inhibition of PARP1, PARP2 and TNKS1 using

commercially available assay kits. Example inhibition curves are shown in Supplementary Figure 2.

Supplementary Figure 2. Example inhibition curves for inhibition of TNKS1 (left panel), PARP1 (middle panel) and

PARP2 (right panel) by TIQ-A. The abscissa shows the inhibitor concentration [M], and the ordinate the enzyme

activity (arbitrary units).

These results were compared to the thermal shifts measured in the DSF screen (main text and

Supplementary Table 3). The resulting correlations are shown in Supplementary Table 4 and Supplementary

Figure 3 below.

TNKS1 PARP1 PARP2

Compound Tm [°C]

Std. Dev [°C]

IC50

[M]

Std. Err.

[M] Tm [°C]

Std. Dev [°C]

IC50

[M]

Std. Err.

[M] Tm [°C]

Std. Dev [°C]

IC50

[M]

Std. Err.

[M]

PJ-34 2.62 0.07 0.570 0.120 7.16 0.03 0.017 0.003 7.70 0.40 0.023 0,008

6(5H)-Phenanthridinone 6.62 0.03 0.054 0.007 6.06 0.13 0.033 0.006 5.29 0.24 0.088 0,040

TIQ-A 5.13 0.20 0.120 0.020 5.40 0.12 0.048 0.007 3.81 0.56 0.210 0,039

106 6.21 0.83 NO FIT 2.36 0.06 0.050 0.011 3.02 0.36 NO FIT

118 5.11 0.24 0.070 0.010 0.96 0.33 NO FIT 0.53 0.18 NO FIT

119 -0.02 0.37 NO FIT 4.93 0.13 0.110 0.030 6.35 0.70 0.120 0.030

Supplementary Table 4. Measured IC50 values for inhibition of TNKS1, PARP1 and PARP2 using commercially

available assay kits.

Supplementary Figure 3. Correlation between thermal stabilization (Tm) and enzyme inhibition potency (pIC50 = -

logIC50) measured using enzymatic assays for TNKS1 (blue diamonds), PARP1 (red squares) and PARP2 (green

triangles). The outlier (compound 106, Tm=2.36°C:pIC50=7.30) was not included in the calculation of the

correlation coefficient.

1x10-8

1x10-7

1x10-6

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

STO814 noZ

1x10-8

1x10-7

1x10-6

0

4000

8000

12000

16000

20000

24000

28000

STO814 noZ

1x10-8

1x10-7

1x10-6

0

40000

80000

120000

160000

200000


8

II. Comparison between Differential Scanning Fluorimetry (DSF) and Surface Plasmon Resonance (SPR) binding

assays to study ligand binding to PARP/ARTD enzymes

Thirteen different compounds identified as binders in the DSF screen were analyzed by SPR for binding to

PARP1, PARP4, TNKS1 and TNKS2 in order to determine dissociation equilibrium constants (KD). For

these compounds, the measured shifts of PARP melting temperatures ranged between 0 and 14 °C.

Supplementary Figure 4 shows example sensorgrams from the SPR analysis, and full results are reported in

Supplementary Tables 5 and 6 below.

For a subset of ligands it was also possible to extract kinetic constants from the SPR measurements

(Supplementary Table 6). These data highlight additional properties of PARP ligand binding that can be

useful to support further optimization/chemical elaboration. For example, slow off-rate is generally

considered to be advantageous to maximize the pharmacological effect of a molecule.

Supplementary Figure 4. Example sensorgrams for the interactions between test compounds and four PARP proteins

obtained by SPR. Compounds at increasing concentrations were injected over the enzyme surface and affinity and

kinetics data were calculated by fitting the sensorgrams to either a steady state or a kinetic interaction model.

Compound names and protein affinities, given as mean ± standard deviation (n=2), are indicated. Insets show the

steady state response levels plotted against compound concentrations and fitted to the 1:1 steady state affinity model.

High affinity binders were evaluated using a 1:1 kinetic model, in which affinity is calculated from the dissociation

and association rate constants (see Supplementary Table 6) ratio. The overlaid dashed lines show the fitted curves.


9

PARP1 PARP4 TNKS1 TNKS2

Compound Tm [°C]

Std.Dev. [°C]

KD

[M]

Std.Dev.

[M] Tm [°C]

Std.Dev. [°C]

KD

[M]

Std.Dev.

[M] Tm [°C]

Std.Dev. [°C]

KD

[M]

Std.Dev.

[M]Tm [°C]

Std.Dev. [°C]

KD

[M]

Std.Dev.

[M]

PJ-34 7.16 0.03 0.045 0.008 3.63 0.25 1.77 0.01 2.62 0.07 13.2 0.4 1.18 0.21 19.8 0.2

6(5H)-Phenanthridinone 6.06 0.13 1.102 0.069 2.59 0.33 11.95 0.95 6.62 0.03 0.74 0.03 4.67 0.52 6.2 0.08

3-Methyl-5-AIQ 5.03 0.10 0.225 0.008 0.87 0.15 43.3 5.7 1.22 0.42 54.7 0.2 0.73 0.06 N/D

5-AIQ 2.87 0.25 1.43 0.03 -0.05 0.50 83.7 0.00 0.33 0.14 >2000 0.06 0.03 N/D

TIQ-A 5.4 0.12 3.62 1.52 1.89 0.06 3.65 0.55 5.13 0.20 0.80 0.07 3.82 0.32 1.04 0.10

98 3.88 0.04 2.46 0.24 2.74 0.38 23.5 1.5 3.6 0.07 16.25 0.05 2.92 0.27 23.4 0.9

105 2.07 0.20 N/D 0.28 0.25 N/D 5.28 0.46 0.53 0.02 4.03 0.28 0.52 0.09

106 2.36 0.06 5.83 0.33 1.37 0.30 N/D 6.21 0.83 1.41 0.02 3.66 0.76 3.36 0.28

ABT-888 9.44 0.09 0.019 0.002 4.59 0.19 N/D 0.28 0.09 >2000 -0.07 0.27 N/D

XAV939 2.87 0.49 1.158 0.033 0.89 0.18 N/D 11.06 0.71 0.044 0.001 7.86 0.79 0.010 0.0002

Olaparib 12.76 0.06 0.002 0.000 7.35 0.10 0.041 0.009 0.67 0.60 12.78 0.48 0.95 0.20 63.850 1.350

118 0.96 0.33 15.40 2.0 0.61 0.04 N/D 5.11 0.24 0.56 0.01 2.56 0.56 0.279 0.002

119 4.93 0.13 0.65 0.11 1.8 0.23 3.00 0.20 -0.02 0.37 >2000 -0.08 0.23 N/D

Supplementary Table 5. Thermal shifts measured by DSF and dissociation equilibrium constants measured

by SPR for ligands binding to different PARP enzymes.

Compound PARP 1 TNKS1 TNKS2

kon (M-1

·s-1

) koff (s-1

) kon (M-1

·s-1

) koff (s-1

) kon (M-1

·s-1

) koff (s-1

)

105

- - (3.3±0.4)×105 (1.8±0.3)×10

-1 (5.8±0.3)×10

4 (3.0±0.3)×10

-2

106

- - (3.0±0.5)×104 (4.3±0.8)×10

-2 - -

XAV939

- - (1.8±0.2)×105 (7.8±0.5)×10

-3 (1.3±0.1)×10

5 (1.3±0.1)×10

-3

ABT-888

(3.2±0.6)×105 (6.0±0.8)×10

-3 - - - -

PJ-34

(3.3±0.3)×105 (1.5±0.2)×10

-2 - - - -

Olaparib

(3.5±0.8)×105 (5.8±0.6)×10

-4 - - - -

TIQ-A (2.0±0.7)×104 (7.2±0.7)×10

-2 - - - -

Supplementary Table 6. Kinetic parameters for the interactions of seven compounds to PARP1, TNKS1 and

TNKS2. Measured values are reported as mean ± standard deviation (n=2).

Interestingly the highly potent tankyrase inhibitor XAV939 (IC50 of 4 and 14 nM for TNKS1 and TNKS2,

respectively19

) displays significant kinetic differences of binding to TNKS1 and TNKS2, respectively (a

five-fold slower off-rate). Since the residues lining the binding pocket are 100% conserved in TNKS1 and

TNKS2, the observed differences could be due to dynamic properties of the protein, possibly involving the

flexibility of the so-called D-loop (main text and Karlberg et al.15

). In the future, dynamic properties of the

D-loop could be approached by methods such as nuclear magnetic resonance (NMR).

Supplementary Figure 5 shows the correlation between thermal shifts and the KD values measured by SPR.

Overall, the correlation between observed thermal shifts and the experimentally determined KD values is

good, and of similar magnitude as previously observed correlations between binding affinity and thermal

shifts (Supplementary Figure 1), and between IC50 values and thermal shifts (Supplementary Figure 3).

Moreover the correlation between KD and reported IC50 values for PARP1 shows a similar pattern

(Supplementary Figure 7).


10

Supplementary Figure 5. Correlation between thermal stabilizations (Tm) and dissociation equilibrium

constants (pKD = - logKD) measured by surface plasmon resonance for TNKS1 (blue diamonds), TNKS2

(red squares), PARP1 (green triangles) and PARP1 (purple crosses).

Supplementary Figure 6. Correlation between measured dissociation equilibrium constants (pKD = -

logKD) by surface plasmon resonance and IC50 values (pIC50 = -logIC50) for PARP1 from enzymatic

assays (Section I above).


11

III. Comparison Differential Scanning Fluorimetry (DSF) and published IC50 values for compounds binding to

PARP1/ARTD1

A literature survey was carried out to extract available PARP1 enzyme inhibition data for previously

published compounds included in the DSF screen. The resulting data, including a comparison with observed

thermal shift measurements, are shown in Supplementary Table 7 and Supplementary Figure 7 below.

Supplementary Figure 7 shows the correlation between thermal shifts observed by us and all available IC50

values.

Compound Tm [°C]

Std. Dev.

Ave. pIC50

Std. Dev.

Ref. 20

Ref. 21

Ref. 22

Ref. 23

Ref. 24

Ref. 25

Ref. 26

Ref. 19

Ref. 27

Ref. 28

Ref. 29

Ref. 30

Ref. 31

This study

pIC50 pIC50 pIC50 pIC50 pIC50 pIC50 pIC50 pIC50 pIC50 pIC50 pIC50 pIC50 pKi pIC50

PJ-34 7.16 0.03 7.73 0.04 7.72 7.70 7.77

4-ANI 4.28 0.28 7.20 0.65 7.66 6.74

ISQ 2.45 0.18 6.82 6.82

DPQ 3.93 0.09 6.03 0.39 6.43 5.66 6.01

5-AIQ 2.87 0.25 5.84 0.13 5.93 5.74

INH2BP 0.67 0.13 5.04 0.05 5.07 5.00

3-AB 0.94 0.11 4.72 0.30 4.89 4.48 5.05 4.45

4-HQN 0.42 1.01 5.23 5.23

6(5H)-Phenan-thridinone 6.06 0.13 6.54 0.69 7.07 5.47 6.52 6.46 6.25 7.48

ABT-888 9.44 0.09 8.28 8.28

TIQ-A 5.40 0.12 6.83 0.68 6.35 7.31

KU0058948 14.95 0.11 8.46 8.46

XAV939 2.87 0.49 5.92 5.92

Olaparib 12.76 0.06 8.30 8.30

Rucaparib 14.35 0.07 8.77 8.77

DR2313 4.26 0.31 6.70 6.70

Supplementary Table 7. Literature data on PARP1 inhibition. Standard deviations were calculated for

those compounds where more than one literature value is available. Note that the value reported for

Rucaparib is the actual inhibition constant. References to original publications are provided in the footnote.


12

Supplementary Figure 7. Correlation between thermal stabilizations (Tm) and PARP1 inhibition potency

(pIC50 = - logIC50) in the available literature.

Summary Overall, as we show for several PARP family members, there is good correlation between observed thermal

shifts obtained using DSF and experimentally determined IC50 values from enzyme inhibition assays.

Moreover, this correlation is of similar magnitude as the observed correlations between binding affinity and

thermal shifts. These observations corroborate the validity of the use of the DSF assay for chemical

profiling of PARP/ARTD inhibitors. Moreover, this conclusion has been reached for other protein classes.32


13


Enzymatic assays. Assay kits (Catalog numbers 80551, 80552, and 80564) were purchased from BPS

Biosciences. Experiments were carried out following the provided protocols. Compounds were dissolved in

DMSO (Sigma-Aldrich D8418). Experimental reactions were set up in duplicate by pre-incubation of the

proteins with compounds in a range between 1nM and 15µM (final concentration of DMSO was 1% in all

samples) for 15min at RT. ADP-ribosylation reactions were then prepared by two-fold dilution into

substrate coated assay plates and incubated in an orbital shaker at 33ºC for 1h at 300rpm.

Chemiluminescence (SuperSignal West Dura; Thermo Fischer Scientific) was detected using a Victor-2

plate reader (PerkinElmer). The resulting data were fitted to a single-site dose-response model using XLFit

(IDBS) to extract experimental IC50 values. The reported errors represent the standard error of the fitted

parameter for each experiment.

Surface Plasmon Resonance. Hexahistidine tagged PARP1, PARP4, TNKS1 and TNKS2 were covalently

immobilized on sensor chip NTA (GE Healthcare) using an amine coupling procedure according to the

manufacturer’s recommendations, with an additional 1-min pulse of 0.5 mM NiCl2 preceding the amine

coupling injections. The enzymes (~20 µg/ml in 20 mM PBS pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05%

surfactant P20) were allowed to bind during 7 min. The resulting immobilization levels of the enzymes

ranged from ~5000 to 8000 resonance units (RU). Measurements were performed on a Biacore™ T200

instrument (GE Healthcare) at 25ºC. The interactions with 13 compounds were analyzed in protein buffer

with the addition of 4% DMSO, using the single cycle kinetics approach with a sequential injection of the

increasing compound concentration over the enzyme surface.33

The concentration series varied from ~1-100

nM to ~1-90 µM using 3-fold dilutions. All measurements were performed in duplicate. After reference and

buffer signal subtraction, the experimental results were fitted to a 1:1 binding model using Biacore™ T200

evaluation software (GE Healthcare).


14

Principal Component Analysis and Multidimensional Scaling Study

A Principal Component Analysis (PCA) was applied on the thermal shifts (Supplementary Table 3) as

obtained from the screening of 185 ligands against the panel of 13 PARP enzymes. Thermal shifts below

1°C were deemed insignificant and were set to 0 to reduce noise. Moreover, non-determined thermal shifts

due to poor quality/missing data points were set to 0 in order to allow the inclusion of these compounds into

the analysis (compound 17 at TNKS1-2 and PARP10; compound 38 at PARP16; compound 185, Rucaparib,

at PARP9). Collectively, the first four principal components derived from the analysis explain

approximately 69% of the variance of the original dataset. Inspection of the contributions (Supplementary

Table 8) and loadings (Supplementary Table 9) of the original variables into the four components reveals

that positive values of the first component (F1, variance 35.3%) encode the average shifts in thermal

stabilities at PARP1, PARP2, PARP3 and PARP4. On the other hand, positive values of the second

component (F2, variance 15.5%) mostly cover the activity of compounds at TNKS1 and TNKS2. Likewise,

positive values of the third component (F3, variance 10.5%) relate to the shifts in thermal stabilities at

PARP9, PARP13 and PARP14, whilst its negative values chart the shifts in thermal stabilities of PARP10.

Positive values of the fourth component (F4, variance 8.2%) indicate stabilization of PARP16, whereas

negative values of this component encode the shifts in thermal stabilities at PARP12 and PARP15

Variable F1 F2 F3 F4

TNKS1 5.35 31.62 0.52 3.31

TNKS2 3.71 31.98 1.75 5.21

PARP1 16.85 3.37 2.45 0.20

PARP2 15.12 5.44 2.35 0.24

PARP3 13.73 3.44 1.70 1.59

PARP4 14.87 2.67 0.85 5.58

PARP16 6.72 0.25 1.36 23.79

PARP13 5.65 1.59 20.09 0.02

PARP12 4.85 6.44 2.86 19.28

PARP9 0.15 0.03 20.55 12.37

PARP15 3.70 0.21 12.11 14.69

PARP14 2.23 5.81 15.82 3.52

PARP10 7.06 7.15 17.57 10.21

Supplementary Table 8. Contributions of the original variables (expressed as %) to the principal

components.


TNKS1 0.231 0.562 0.072 -0.182

TNKS2 0.193 0.566 0.132 -0.228

PARP1 0.410 -0.183 0.157 -0.045

PARP2 0.389 -0.233 0.153 -0.049

PARP3 0.371 -0.185 0.130 -0.126

PARP4 0.386 -0.164 0.092 0.236

PARP16 0.259 -0.050 0.117 0.488

PARP13 -0.238 -0.126 0.448 0.015

PARP12 0.220 -0.254 -0.169 -0.439

PARP9 -0.039 0.018 0.453 -0.352

PARP15 0.192 -0.045 -0.348 -0.383

PARP14 0.149 0.241 0.398 0.187

PARP10 0.266 0.267 -0.419 0.320

Supplementary Table 9. Loadings of the original variables into the principal components.

Inspection of the correlation coefficients between the original variables and the aforementioned principal

components shows that only the first two components are in fair correlations (R2 > 0.75) with the respective

original variables that present the highest contributions (Supplementary Table 10). This observation


15

suggests that overall the dataset of compounds tested in the panel of PARPs covers a good range of

activities at PARP1-4 (F1) and TNKS1-2 (F2), with minor and/or no activities at the remaining PARP

enzymes.


TNKS1 0.50 0.80 0.08 -0.19

TNKS2 0.41 0.80 0.15 -0.24

PARP1 0.88 -0.26 0.18 -0.05

PARP2 0.83 -0.33 0.18 -0.05

PARP3 0.79 -0.26 0.15 -0.13

PARP4 0.83 -0.23 0.11 0.24

PARP16 0.56 -0.07 0.14 0.50

PARP13 -0.51 -0.18 0.52 0.02

PARP12 0.47 -0.36 -0.20 -0.45

PARP9 -0.08 0.02 0.53 -0.36

PARP15 0.41 -0.06 -0.41 -0.40

PARP14 0.32 0.34 0.46 0.19

PARP10 0.57 0.38 -0.49 0.33

Supplementary Table 10. Correlation coefficients (R2) between original variables and principal

components.

Although the third and fourth components are not strongly correlated with stabilization of specific PARPs,

inspection of Supplementary Figure 8 may offer clues to drive chemical biology and medicinal chemistry

towards subtype specific PARP inhibitors (see main text for further discussion).

Supplementary Figure 8. Activity space of PARP inhibitors according to the third and fourth components.

Selected PARP inhibitors in clinical phase study as well as inhibitors commonly used as chemical tools are

labeled. Compound 148 is also labeled, lying at the edge of negative values of F3 and positive values of F4.


16

Recent phylogenetic studies have classified members of the PARP superfamily into six clades that have

evolved from two ancestral genes: the first endowed with poly-ADP-ribosylation activity and involved in

DNA repair and genome integrity; the second acting in mono-ADP-ribosylation and involved in the

regulation of signalling pathways and gene expression.34

Mammalian members of PARP superfamily covers

five of the six clades, with PARP1-3 belonging to clade I; PARP7, PARP9, PARP10-15 grouping into

different subgroups of clade III; TNKS1-2 being members of clade IV; clade V comprising PARP4; and

PARP6, PARP8 and PARP16 being grouped in clade VI. In order to gain insights into ligand-based

relationships among PARPs used in this study, a Multidimensional Scaling (MDS) was carried out. The aim

of this method is to build a mapping of the enzymes from a similarity matrix composed of Pearson

correlation coefficients calculated using the average thermal shifts of the dataset (PARP activity space). To

build an optimal representation, the MDS algorithm minimizes a criterion called "stress"; the closer the

stress to zero, the better the representation. The MDS map of PARPs is shown in Supplementary Figure 9,

whilst Supplementary Table 11 reports the distances between couples of enzymes as measured in the map.

High distances indicate remote or no relationships, and low distances suggest closer relationships.

From the inspection of Supplementary Figure 9, two groups of closely related enzymes stand out: the first is

composed of TNKS1 and TNKS2, resembling clade IV; the second is made of PARP1-4, suggesting similar

requirements on part of catalytic clefts of these enzymes of clade I and V to bind small molecule inhibitors.

Supplementary Figure 9. MDS map of PARPs according to the activity space. Stress = 0.206

TNKS2

TNKS1

PARP1

PARP2

PARP3

PARP4PARP12

PARP15PARP16

PARP9

PARP14

PARP13

PARP10

TNKS2

TNKS1

PARP1

PARP2

PARP3

PARP4PARP12

PARP15PARP16

PARP9

PARP14

PARP13

PARP10


17

TNKS1 TNKS2 PARP1 PARP2 PARP3 PARP4 PARP16 PARP13 PARP12 PARP9 PARP15 PARP14 PARP10

TNKS1 0 0.075 0.723 0.788 0.806 0.622 0.663 1.434 1.155 0.928 1.130 0.465 0.441

TNKS2 0.075 0 0.782 0.843 0.867 0.676 0.681 1.410 1.199 0.880 1.156 0.426 0.516

PARP1 0.723 0.782 0 0.103 0.093 0.131 0.480 1.385 0.503 1.197 0.649 0.838 0.510

PARP2 0.788 0.843 0.103 0 0.128 0.167 0.444 1.318 0.404 1.176 0.551 0.850 0.609

PARP3 0.806 0.867 0.093 0.128 0 0.224 0.558 1.445 0.476 1.283 0.667 0.930 0.555

PARP4 0.622 0.676 0.131 0.167 0.224 0 0.367 1.291 0.549 1.070 0.626 0.707 0.489

PARP16 0.663 0.681 0.480 0.444 0.558 0.367 0 0.932 0.612 0.735 0.482 0.482 0.760

PARP13 1.434 1.410 1.385 1.318 1.445 1.291 0.932 0 1.225 0.655 0.901 0.994 1.673

PARP12 1.155 1.199 0.503 0.404 0.476 0.549 0.612 1.225 0 1.293 0.324 1.093 1.013

PARP9 0.928 0.880 1.197 1.176 1.283 1.070 0.735 0.655 1.293 0 1.038 0.466 1.283

PARP15 1.130 1.156 0.649 0.551 0.667 0.626 0.482 0.901 0.324 1.038 0 0.939 1.114

PARP14 0.465 0.426 0.838 0.850 0.930 0.707 0.482 0.994 1.093 0.466 0.939 0 0.825

PARP10 0.441 0.516 0.510 0.609 0.555 0.489 0.760 1.673 1.013 1.283 1.114 0.825 0

Supplementary Table 11. Distances measured in the MDS map of the PARP/ARTD superfamily.

Other PARPs occupy distinct and remote regions of the MDS map, with PARP9 being the most distantly

located enzyme. Interestingly, the remote distance of PARP9 is in agreement with its catalytic “diversity”.

Indeed, PARP9 has been shown to lack poly or mono-ADP-ribose transferase activity, missing several key

residues within the catalytic cleft.35

The MDS map shows the formation of two clusters composed of TNKS1-2 and PARP1-4, respectively.

This is in agreement with the aforementioned analysis of the activity space and suggests how the dataset of

this study may be biased by compounds showing a good range of activities at PARP1-4 and TNKS1-2, but

minor or no activities towards the remaining enzymes.

As a consequence, similar to other studies reporting no linear relationship between sequence similarity and

small molecule inhibition of kinases,36

we observe here a general trend where compounds stabilizing one

PARP of clade I are very likely to show activity towards other PARPs from the same clade. Furthermore,

there is still a good probability that compounds acting at clade I may stabilize PARP4 of clade V. Outside

clade I and clade V, while in clade IV it is almost reliable that compounds acting at TNKS1 are also able to

stabilize TNKS2, it is less frequent that they are also active towards other clades, although some exceptions

are present in the dataset (e.g., Rucaparib, TIQ-A and 6(5H)Phenanthridinone).


18

Structural studies of TNKS2 and PARP14 using X-ray crystallography

Observed binding of compounds to the NAD+ binding site

All compounds bind in the NAD+ co-substrate binding site. There are a number of conserved interactions

comparing with other PARP inhibitor complexes present. Typically, a tyrosine residue makes stacking

interactions with the ligand, and a serine hydroxyl and main chain atoms from a glycine form hydrogen

bonds with the groups, mimicking the carboxamidyl of the nicotinamide moiety of NAD+.

TNKS2 – 106: The Tyr1071 side chain stacks with the ring system. The Ser1068 hydroxyl and the Gly1032

main chain oxygen and nitrogen form hydrogen bonds with the (NAL-nitrogen), the (NAK-nitrogen) and

the substituted carbonyl (OAB) on the naphtyridine ring. The D-loop has a different conformation compared

to previously determined TNKS2 structures; the Tyr1050 side chain has flipped out of the pocket while

Ile1051 is pointing inwards. As a result the pocket is slightly more open and can accommodate compound

106, which has a methyl group that would otherwise clash with Tyr1050 (Supplementary Figure 10a).

TNKS2 – 183: The Tyr1071 side chain stacks with the ring system. The Ser1068 hydroxyl and the Gly1032

main chain oxygen and nitrogen form hydrogen bonds to the (NAI-nitrogen), to the (N1-nitrogen) and to the

substituted carbonyl (O6) on the pyrimidine ring. The nitrogen on the outer ring (NAI) hydrogen bonds via

a water molecule with the main chain oxygen of His1048 and the mainchain nitrogen of Tyr1060

(Supplementary Figure 10b).

TNKS2 – ACT20400: The Tyr1071 side chain stacks with the ring system. The Ser1068 hydroxyl and the

Gly1032 main chain oxygen and nitrogen form hydrogen bonds with the (NAI-nitrogen) and with the

substituted carbonyl on the pyrazine ring. The bromine makes interactions with two waters, one which

bridges to the hydroxyl on Ser1033. The side chain of His1031 is in the same network of hydrogen bonds. A

few residues make hydrophobic interactions, namely Tyr1050, Tyr1060, Ala1062 and Lys1067

(Supplementary Figure 10c).

TNKS2 – ACT24188: The Tyr1071 side chain stacks with the ring system. The Ser1068 hydroxyl and the

Gly1032 main chain oxygen and nitrogen form hydrogen bonds with the (NAN-nitrogen) and with the

substituted carbonyl on the pyrimidine ring. The second nitrogen (NAT) on the central ring hydrogen bonds

via a water molecule with the main chain oxygen of His1048 and the main chain nitrogen of Tyr1060. The

Tyr1050, Tyr1060, Phe1035 and Ile1075 side chains are involved in hydrophobic interactions with the

ligand (Supplementary Figure 10d).

TNKS2 – ACT18395: The Tyr1071 side chain stacks with the ring system. The Ser1068 hydroxyl forms a

hydrogen bond with the (NAK-nitrogen), and the Gly1032 main chain oxygen with the (NAN-nitrogen).

Notably, an amide group that in other complexes binds to conserved Gly-Ser motif, is replaced here by two

nitrogens. Potentially, this novel chemotype could be used as a new starting point for a different compound

class. Further, the two nitrogens on the pyridazine ring interact via a water molecule that bridges to the side

chain of Glu1138. There are a number of hydrophobic interactions, in particular at the vicinity of the

binding pocket, involving Tyr1050, Tyr1060, Phe1061, Ala1062, Pro1034, Phe1035 and Ile1075

(Supplementary Figure 10e).

PARP14 – 19: The Tyr1633 and Tyr1646 side chains stack with the ring. The Ser1641 hydroxyl and the

Gly1602 main chain oxygen and nitrogen form hydrogen bonds with the amide group. The hydroxyl of

Tyr1646 bonds with the amino group of 3-aminobenzamide (Supplementary Figure 10f).


19

PARP14 – 8: The Tyr1633 and Tyr1646 side chains stack with the ring system. The Ser1641 hydroxyl and

the Gly1602 main chain oxygen and nitrogen form hydrogen bonds with the amide group (Supplementary

Figure 10g).

PARP14 - 98: The Tyr1633 and Tyr1646 side chains stack with the ring system. The Ser1641 hydroxyl and

the Gly1602 main chain oxygen and nitrogen form hydrogen bonds with the amide group. Tyr1620 stacks

with the triazole-ring, and forms a hydrogen bond with one of the nitrogens on the ring using its backbone

oxygen (Supplementary Figure 10h).

PARP14 - 145: The Tyr1633 and Tyr1646 side chains stack with the ring system. The Ser1641 hydroxyl

and the Gly1602 main chain oxygen and nitrogen form hydrogen bonds with the amide group

(Supplementary Figure 10i).


20

Supplementary Figure 10. Details of the active sites in TNKS2 and PARP14 protein:ligand complexes. Electron

densites of the ligands were contoured at 1.0 in a 2Fobs-Fcalc map. TNKS2 in complex with compounds (a) 106

(ENAMINE, T598968), (b) 183 (Maybridge, RF03877), (c) ACT20400 (CHEMDIV, C800-1144), (d) ACT24188

(CHEMDIV, F019-2529) and (e) ACT18395 (CHEMDIV, 8233-1036). PARP14 in complex with (f) 19 (3-

aminobenzamide), (g) 8 [6(5H)-Phenanthridinone], (h) 98 (ENAMINE, T5867921) and (i) 145 (CHEMDIV, 3909-

7439).

a b c

d e f

g h i


21

Supplementary Table 12. Data collection, phasing and refinement statistics for TNKS2 ligand complexes.

* Values in parentheses are for the outermost resolution shell. a

Rmerge = III /| | , where I is the intensity measurement for a given reflection and I is the average intensity for

multiple measurements of this reflection. b

R = ||/|||||| obscalcobs FFF , where Rfree is calculated for a randomly chosen 5-10% of reflections, which were not

used for structure refinement, and Rall is calculated for all reflections. c The Ramachandran plot was calculated by Molprobity server (http://molprobity.biochem.duke.edu/).

Ligand 106 183 ACT20400 ACT24188 ACT18395

Data collection

Synchrotron ESRF ESRF ESRF Bruker BESSY

Beam line ID-29 ID-29 ID-29 Proteum X8 BL14-2

Wavelength (Å) 0.97908 0.97908 0.97908 1.54177 0.91841

Space group C2221 P3121 C2221 C2221 C2221

Unit cell

dimensions, a, b, c

(Å)

90.55, 97.73,

119.13

92.48, 92.48, 53.51 91.30, 98.21,

118.68

90.65, 97.89,

118.21

91.02, 97.70,

118.89

α, β, γ (°) 90, 90, 90 90, 90, 120 90, 90, 90 90, 90, 90 90, 90, 90

Resolution (Å) 45.2 -1.80 (1.85 –

1.80)

46.2 – 2.30 (2.36 –

2.30)

45.0 – 1.90 (1.95 –

1.90)

66.5 – 2.50 (2.60 –

2.50)

35.0 – 1.90 (1.95 –

1.90)

Unique reflections 49107 (3582) 11932 (880) 42208 (3067) 18543 (1813) 41865 (2963)

R-merge (%)a 5.4 (45.2) 9.7 (55.7) 6.2 (18.3) 18.6 (38.6) 6.5 (53.0)

Completeness (%) 99.9 (99.8) 99.1 (97.8) 99.9 (99.7) 99.0 (91.5) 99.6 (96.9)

Redundancy 7.1 (5.0) 10.9 (10.5) 7.2 (6.9) 6.6 (4.0) 6.1 (4.8)

<I>/<I> 24.6 (3.7) 22.2 (5.9) 23.7 (10.2) 7.6 (2.5) 20.9 (3.5)

Phasing

MR starting model 3kr7 3kr7 3kr7 3kr7 3kr7

Refinement

Resolution (Å) 45.2 – 1.80 46.2 – 2.30 45.0 – 1.90 50.0 – 2.50 35.0 – 1.90

R-all (%)b 17.39 17.95 16.14 19.67 17.41

R-free (%)b 20.56 22.73 19.50 26.13 20.45

r.m.s.d. bond

length (Å)

0.018 0.012 0.019 0.010 0.019

r.m.s.d. bond angle

(°)

1.7 1.3 1.7 1.1 1.7

Ramachandran plot

c

Most favoured (%) 98.3 98.6 98.3 96.5 98.8

Allowed (%) 100 100 100 100 100


22

Supplementary Table 13. Data collection, phasing and refinement statistics for PARP14 ligand complexes.

* Values in parentheses are for the outermost resolution shell. a

Rmerge = III /| | , where I is the intensity measurement for a given reflection and I is the average intensity for

multiple measurements of this reflection. b

R = ||/|||||| obscalcobs FFF , where Rfree is calculated for a randomly chosen 5-10% of reflections, which were not

used for structure refinement, and Rall is calculated for all reflections. c The Ramachandran plot was calculated by Molprobity server (http://molprobity.biochem.duke.edu/).

Ligand 19 8 98 145

Data collection

Synchrotron DIAMOND BESSY BESSY ESRF

Beam line I03 BL14-2 BL14-1 ID-29

Wavelength (Å) 0.98003 0.91841 0.91841 0.97908

Space group C2 P6522 P212121 P212121

Unit cell

dimensions, a, b, c

(Å)

82.75, 144.27,

79.70

82.37, 82.37,

434.48

40.57, 66.42,

145.15

39.47, 67.76,

144.80

α, β, γ (°) 90, 100.55, 90 90, 90, 120 90, 90, 90 90, 90, 90

Resolution (Å) 25.0 – 2.80 (2.87 –

2-80)

35.0 – 2.30 (2.36 –

2.30)

35.0 – 2.40 (2.46 –

2.40)

35.0 – 1.50 (1.54 –

1.50)

Unique reflections 22380 (1657) 40412 (2896) 16003 (1155) 62739 (4516)

R-merge (%)a 14.6 (75.4) 16.6 (73.2) 10.9 (50.5) 7.0 (39.1)

Completeness (%) 98.7 (98.0) 99.9 (100) 99.8 (99.9) 99.1 (97.3)

Redundancy 6.6 (6.6) 13.5 4.4 (4.5) 6.4 (6.4)

<I>/<I> 10.2 (2.9) 15.7 (3.5) 12.6 (3.8) 17.6 (5.8)

Phasing

MR starting model 3blj 3se2 3se2 3se2

Refinement

Resolution (Å) 24.6 – 2.80 34.6 – 2.30 33.7 – 2.40 34.7 – 1.50

R-all (%)b 25.50 21.01 21.37 18.21

R-free (%)b 29.20 25.45 25.23 21.23

r.m.s.d. bond

length (Å)

0.007 0.012 0.028 0.018

r.m.s.d. bond angle

(°)

0.96 1.3 2.1 1.7

Ramachandran

plot c

Most favoured (%) 91.4 97.3 97.9 98.4

Allowed (%) 99.4 99.7 100 100


23


Protein Production

Human PARP14 catalytic domain was obtained as a synthetic gene. The region encoding PARP14 residues

1530-1720 was subcloned into expression vector pNIC-Bsa4 by ligation-independent cloning, adding an N-

terminal hexahistidine tag and a TEV-protease cleavage site (MHHHHHHSSGVDLGTENLYFQ*SM,

where * indicates the site of cleavage).37

The cDNA coding for human TNKS2 were obtained from Origene (accession code NM_025235). The

sequences encoding residues 946-1162 and 952-1166 for TNKS2 were subcloned into expression vector

pNIC-CH (adding a C-terminal hexahistidine tag without TEV-protease cleaving site) or into pNIC-Bsa4.

Recombinant expression in Escherichia coli and purification of proteins followed the previously published

protocol for TNKS1.38

Purified N-terminally hexahistidine tagged TNKS2 protein was difficult to concentrate using an Amicon

Ultra-15 centrifugal filter device (Millipore, 10,000 MWCO). Chymotrypsin (1:100 w/w) was added at low

TNKS2 concentration, and the protein was concentrated to 13.7 mg/ml. Mass spectrometry identified two

chymotrypsin cleaved TNKS2 fragments (TNKS2947-1113

and TNKS2947-1162

). Purified TNKS2 C-terminal

hexahistidine tagged protein concentrated without problem to 11 mg/ml.

Purified PARP14 was incubated with hexahistidine tagged TEV-protease (1:20 ratio) and further purified.

Both uncleaved and cleaved PARP14 were used in crystallization.

Screening using Corning Epic technology

An exploratory study was carried out using the Corning Epic technology.39

In this study we screened 10000

compounds for binding to TNKS1. Briefly, 50µg/ml TNKS1 was immobilized for one hour at 25°C in an

Epic biochemical plate. To identify non-specific and promiscuous compounds, 75µg/ml streptavidin (in

20mM sodium acetate pH 5.1) was immobilized for one hour. The plates were subsequently washed three

times with 100mM Tris-HCl pH 7.4, binding buffer (PBS pH 7.4 and 0.2% DMSO) was added and plates

were incubated in the EPIC carousel at 26°C for 4 hours. A baseline read was recorded prior to addition of

15µl of compound (10µM final concentration in presence of 0.2% SDS). Binding was recorded after 25

minutes of equilibration. Hits were verified using DSF, and co-crystallization trials were conducted with

TNKS1 and TNKS2. To date, this project has yielded high resolution structures of TNKS2 in complex with

three novel ligands (ACT20400, ACT24188 and ACT18395) reported herein.

Crystallization

Crystals of TNKS2 apo crystals were obtained by sitting drop vapor diffusion at 4 ºC in droplets consisting

of 0.1 µl protein solution (13.7 mg/ml) and 0.2 µl of well solution (0.1 M Tris-HCl pH 8.5, 20-25 %

PEG3350, and 0.2 M of either lithium sulfate or ammonium sulfate).

Crystals of the TNKS2–compound complexes (compounds 106, ACT20400, ACT24188 and ACT18395)

were obtained by addition of 0.1 μl of a 5-10 mM solution of compound dissolved in DMSO to drops

containing crystals grown as described above, and incubating at 4 ºC for 7-10 days. Crystals were

transferred to cryoprotectant solution (well solution supplemented with 25 % glycerol and 0.2 M NaCl) and

flash frozen in liquid nitrogen.


24

Crystals of TNKS2 in complex with 183 (Maybridge, RF03877) were grown using vapor diffusion at 4°C

by mixing 0.2 μl of protein solution at 7.1 mg/ml including 183 (5:1 molar ratio) and 0.1 μl reservoir

solution containing 1.34 M K2H-phosphate, 0.06 M NaH2-phosphate. Crystals appeared after two weeks and

continued to grow for another 6 weeks. Crystals were transferred to cryoprotectant solution (1.2 M

KH2PO4, 30% Glycerol, 0.2 M NaCl) and frozen in liquid nitrogen.

Crystals of PARP14 in complex with 19 (3-aminobenzamide) were grown using vapor diffusion at 20°C by

mixing 0.1 μl of protein solution (uncleaved batch) at 30 mg/ml including 19 (5 mM) and 0.1 μl reservoir

solution containing 20 % PEG3350, 0.2 M sodium malonate. Crystals appeared after one month and were

transferred to a cryoprotectant solution (well solution supplemented with 20 % PEG400 and 0.2 M NaCl)

and frozen in liquid nitrogen.

Crystals of PARP14 in complex with 8 (6(5H)-Phenanthridinone) were grown using vapor diffusion at 20°C

by mixing 0.1 μl of protein solution (cleaved variant) at 39 mg/ml including 19 (10 mM) and 0.1 μl

reservoir solution containing 20% PEG3350, 0.2M potassium thiocyanate, 0.1 M Bis-tris-propane pH 7.5.

Crystals appeared after one month and were soaked with 8 for 20 h. They were further transferred to a

cryoprotectant solution (well solution supplemented with 20 % glycerol and 0.2 M NaCl) and frozen in

liquid nitrogen.

Crystals of PARP14 in complex with 98 (ENAMINE, T5867921) were grown using vapor diffusion at 20°C

by mixing 0.2 μl of protein solution (cleaved variant) at 35 mg/ml including 98 and 0.1 μl reservoir solution

containing 25 % PEG3350, 0.2 M sodium nitrate. Crystals appeared after one month and were transferred to

a cryoprotectant solution (well solution supplemented with 15 % glycerol and 0.2 M NaCl) and frozen in

liquid nitrogen.

Crystals of PARP14 in complex with 145 (CHEMDIV, 3909-7439) were grown using vapor diffusion at

20°C by mixing 0.2 μl of protein solution (cleaved variant) at 17 mg/ml including 145 and 0.1 μl reservoir

solution containing 20 % PEG3350, 0.1 M Bis-tris pH 6.5, 0.2 M sodium sulfate. Crystals appeared after

one month and were transferred to a cryoprotectant solution (well solution supplemented with 20% glycerol

and 0.2M NaCl) and frozen in liquid nitrogen.

Data collection, structure solution, and refinement

Diffraction data for the different complexes were collected at ESRF (Grenoble, France), BESSY (Berlin,

Germany), DIAMOND (Oxfordshire, UK) or on an X8 PROTEUM system equipped with a four-circle

Kappa goniostat and a PLATINUM-135 CCD (Bruker AXS, Delft, Netherlands). All data were indexed and

integrated using standard protocols, solved by molecular replacement and refined using previously

described procedures.40

For further details on data processing, phasing and refinement statistics, see

Supplementary Tables 12 and 13. All structures have been deposited to the Protein data bank with

accession codes 3GOY, 3MHJ, 3MHK, 3P0N, 3P0P, 3P0Q, 3SE2, 3SMI and 3SMJ. Figures were generated

using PyMOL (The PyMOL Molecular Graphics System, Version 0.99, Schrödinger, LLC.).


25

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