Applications of NMR screening techniques to the pharmaceutical target Checkpoint kinase 1

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Applications of NMR screening techniques to the pharmaceutical target Checkpoint kinase 1 N. Lancelot a,∗ , M. Piotto b,∗∗ , I. Theret c , B. Lesur c , P. Hennig a a b c

Institut de Recherches Servier, Analytical and Physical Chemistry Department, 11 rue des Moulineaux, 92150 Suresnes, France Bruker BioSpin, Laboratoire d’applications RMN, 34 rue de l’industrie, 67166 Wissembourg, France Institut de Recherches Servier, Chimie Partenariats et Modélisation Moléculaire, 125 Chemin de Ronde, 78290 Croissy-Sur-Seine, France

a r t i c l e

i n f o

Article history: Received 4 July 2013 Received in revised form 17 October 2013 Accepted 21 October 2013 Available online xxx Keywords: Checkpoint kinase 1 NMR screening Group epitope mapping Saturation Transfer Difference Transferred NOESY

a b s t r a c t Ligand screening techniques based on NMR spectroscopy are not as sensitive as other commonly used methods like fluorescence, radiolabeling and surface plasmon resonance. However, using modern NMR instrumentation, they can achieve reliable screening under near physiological condition using as little as 4.6 nmol of receptor and 100 nmol of ligand. Additionally, these NMR methods can also provide valuable and specific information on the ligand under investigation such as the dissociation constant KD , the binding epitope and most importantly some structural information on the actual conformation in the bound state. In this manuscript, we describe the use of NMR based screening techniques (“Saturation Transfer Difference” (STD) and “Water Ligand Observed via Gradient SpectroscopY” (WaterLOGSY)) to detect small therapeutic molecules that interact with the DNA damage checkpoint enzyme Checkpoint kinase 1 (Chk1). After the identification of the most potent ligand, we used specific NMR experiments to perform the epitope mapping of this ligand (“Group epitope mapping-STD” (GEM-STD), “Difference of Inversion REcovery rate with and without Target IrradiatiON” (DIRECTION)) and to characterize its bound conformation (“Transferred-Nuclear Overhauser Effect SpectroscopY” (tr-NOESY), “Transferred-Rotating frame Overhauser Effect SpectroscopY” (tr-ROESY)). Finally, we used molecular docking procedures to position the ligand within the active site of Chk1. On the experimental level, a comparison between NMR studies performed in a 90%H2 O/10%D2 O buffer and a 100% D2 O buffer is also presented and discussed. © 2013 Elsevier B.V. All rights reserved.

1. Introduction To protect the integrity of their DNA against attacks from various endogenous and environmental sources, cells have evolved a genome surveillance network that carefully coordinates DNA repair with cell cycle progression. DNA double strand breaks (DSBs) are considered the most toxic type of DNA damage. If left unrepaired or repaired improperly, they cause chromosomal aberrations, which

Abbreviations: AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; Chk1, Checkpoint kinase 1; DIRECTION, Difference of Inversion REcovery rate with and without Target IrradiatiON; DSBs, double strand breaks; HEPES, 4-(2HydroxyEthyl)-1-PiperazineEthaneSulfonic acid; HTS, high throughput screening; MPF, maturation promoting factor; PMSF, phenylmethylsulfonyl fluoride; STD, Saturation Transfer Difference; GEM-STD, Group epitope mapping-STD; STDTOCSY, Saturation Transfer Difference-TOtal Correlation SpectroscopY; Tr-NOESY, Transferred-Nuclear Overhauser Effect SpectroscopY; Tr-ROESY, TransferredRotating frame Overhauser Effect SpectroscopY; WaterLOGSY, Water Ligand Observed via Gradient SpectroscopY. ∗ Corresponding author. Tel.: +33 1 55 72 21 11. ∗∗ Corresponding author. Tel.: +33 3 88 73 68 62. E-mail addresses: [email protected] (N. Lancelot), [email protected] (M. Piotto).

may be lethal or result in oncogenic transformation. A prominent cellular response to DSBs is the focal assembly of a large number of DNA repair proteins and checkpoint proteins at the site of damage. At the site of DNA damage, “mediator” proteins are in charge of recruiting “signal transducers” to molecules “sensing” the damage. Chk1 and Chk2 are two kinases which are signal transducers in the regulation of cellular cycle and in the response to DNA damages. These proteins are Ser/Thr protein kinases with a moderate specificity of substratum. Their target is the machinery of the cell cycle in order to inhibit its spread. In mammalian cells, the signal induced by DSBs is detected by ATM and transmitted to Chk2 whereas the signal induced by UV radiations is detected by ATR and transmitted to Chk1. Chk1 is unstable (half-life lower than 2 h) and is only expressed in S and G2 phases. This kinase is always activated even during normal cellular cycle but is more activated faced with damages or replication arrest. This is a kinase that phosphorylates Cdc25, an important phosphatase in cell cycle control, particularly for entry into mitosis. Cdc25, when phosphorylated on serine 216 by Chk1 creates a binding site for 14-3-3 protein and thus becomes bound by an adaptor protein in the cytoplasm. Therefore it is inhibited from removing the inhibiting phosphate from MPF (mitotic/maturation promoting factor) added by Wee1.

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Consequently, a cell is prevented from entering mitosis. Thus, in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thereby preventing the activation of the Cdc2-cyclin B complex and mitotic entry. Chk1 is a 56 kDa protein kinase which has a C-terminal catalytic domain and a domain rich in SQ/TQ phosphorylation sites. Molecules inhibiting Chk1 have been tested in the treatment of cancer [1–4]. Contrary to normal cells, cancerous cells are more sensitive to inhibitors. The inhibitors will be thus more effective on tumoral cells. Chk1 inhibitors have been targeted as possible chemo- and radio-potentiators [5–10]. Most of published Chk1 inhibitors come from hits discovered by high throughput screening (HTS) of large compound libraries [11–13]. In this manuscript, we apply NMR based screening techniques [14,15] to characterize small therapeutic molecules that inhibit the catalytic domain of DNA damage checkpoint enzyme Chk1 (Checkpoint kinase 1). 2. Materials and methods 2.1. Expression and purification of Chk1 2.1.1. Subcloning The subcloning was described in Ferry et al. [16].

further developed with 100 ml of buffer H containing Tris–HCl (25 mM), NaCl (125 mM), EDTA (1 mM), glycerol (10%), isopropanol (30%) (pH* 8). Fractions containing Chk1-cat-HT were pooled and loaded onto a filtering gel HiLoad 26/60 Superdex 75 pg column equilibrated in buffer I containing HEPES (25 mM), NaCl (500 mM), EDTA (1 mM), DTT (5 mM), Glycerol (10%), octylglucoside (0.05%) (pH* 7.5). Elution was performed by addition of buffer I. The fractions of interest were then analyzed by SDS-PAGE and fractions containing Chk1-cat-HT were pooled and concentrated 5.6 times on an amicon system on a PES membrane (threshold cut of 5 kDa). The concentration was calculated from the results of absorbance at 280 nm, the theoretical protein molecular mass (33,820 Da) and the theoretical His-Chk1 (1–289) epsilon (47,900 M−1 cm−1 ). The 20 mL concentrated Chk1-cat-HT protein were then dialysed against buffer J containing HEPES (25 mM), NaCl (500 mM), EDTA (1 mM), DTT (5 mM), deuterated glycerol (10%), n-Octylglucoside (0.05%) (pH* 7.5) in a dialysis tube (threshold cut of 12–14 kDa) during 3 h at 4 ◦ C. The dialysis buffer was changed twice after 4 h and the last dialysis lasted 12.5 h. Aliquots of protein were frozen at −20 ◦ C. The final sample was determined to be 89% pure by densitometric scanning. The Chk1 protein used in this study has a molecular weight of 33.8 kDa and contains a total of 289 amino acids. 2.2. NMR experiments

2.1.2. Chk1-cat-HT (C-terminal His-tagged Chk1 1–289) protein overproduction and purification Overproduction of pGTPB302 Chk1cat His (1–289) was achieved in Sf9 cells with a MOI of 5 for 48 h at 27 ◦ C. Cells were harvested by centrifugation and resuspended in 800 mL of cold lysis buffer containing HEPES (50 mM) (4-(2HydroxyEthyl)-1-PiperazineEthaneSulfonic acid)–NaCl (100 mM) – 2-mercaptoethanol (10 mM) – Triton X-100 (0.1%) – benzamidine (1 mM) – AEBSF (1 mM) (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) – PMSF (1 mM) (phenylmethylsulfonyl fluoride) (pH* 7.5). The suspension was homogenized (Ultra-Turrax, 8000 rpm for 30 s) and cells were sonicated 2 times for 30 s with pulses of 0.5 s at 100% power. Soluble and non-soluble fractions were recovered by centrifugation at 20,000 × g during 30 min at 4 ◦ C. All subsequent purification procedures were performed at 4 ◦ C using an Akta Purifier. The soluble fraction was diluted twice in Buffer A containing HEPES (50 mM), benzamidine (1 mM), AEBSF (0.1 mM), PMSF (1 mM) (pH* 7.5) and loaded onto a 400 mL Q-Sepharose FF column and equilibrated with buffer B containing HEPES (50 mM), Triton X-100 (0.05%), NaCl (1 M) (pH* 7.5) then with buffer C containing HEPES (50 mM), Triton X-100 (0.05%), NaCl (100 mM) (pH* 7.5). Elution was performed by addition of buffer B. The fractions of interest were then analyzed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Chk1-cat-HT was recovered between 0 and 40% of buffer B, fractions were pooled and loaded onto a 10 mL Ni-TED column and equilibrated in buffer D containing HEPES (50 mM), Triton X-100 (0.05%), NaCl (500 mM) (pH* 7.5). The column was developed with first 100% elution buffer D, then 100% elution buffer E containing HEPES (50 mM), Triton X-100 (0.05%), NaCl (500 mM), imidazole (500 mM) (pH* 7.5) and further with 100% elution buffer D. Active fractions were fractions 2 to 6 of the elution process. Fractions containing Chk1-cat-HT were pooled, diluted twice with buffer F(2X) containing Tris–HCl (50 mM), NaCl (250 mM), EDTA (2 mM), DTT (10 mM), glycerol (20%), Ammonium sulfate (1.6 mM) (pH* 8) and loaded onto a 20 mL Resource 15PHE column equilibrated in buffer F containing Tris–HCl (50 mM), NaCl (250 mM), EDTA (2 mM), DTT (10 mM), glycerol (20%), Ammonium sulfate (0.8 mM) (pH* 8). The column was developed with a gradient from 100% buffer F to 100% buffer G containing Tris–HCl (25 mM), NaCl (125 mM), EDTA (1 mM), DTT (5 mM), glycerol (10%) (pH* 8), and

Samples used for the NMR experiments were prepared using a solution containing 23 ␮M of Chk1 in HEPES buffer in 100% H2 O containing HEPES (25 mM), NaCl (500 mM), EDTA (1 mM), DTT (5 mM), octylglucoside (0.05%), deuterated glycerol (10%) (pH* 7.5) and a solution containing the ligand at a concentration of about 10.5 mM in DMSO-d6 . The NMR tubes were prepared by adding 200 ␮l of the Chk1 solution, 10 ␮l of the inhibitor solution and 20 ␮l of D2 O to a 3 mm NMR tube. Typically, the final tube contained 20 ␮M of Chk1 and about 500 ␮M of ligand which corresponds roughly to about 155 ␮g (4.6 nmol) of protein and 43 ␮g of ligand (104 nmol). The use of 3 mm NMR tubes, instead of the classical 5 mm tubes, is particularly interesting in this type of study since it allows to minimize the amount of protein used and to reduce salt effects induced by the buffer (lengthening of the 90◦ 1 H pulse and corresponding loss of sensitivity of the NMR experiment). In order to obtain the protein in a 100% D2 O buffer, the Chk1 protein was dialysed (Slide-a-Lyser (Pierce) threshold cut of 10 kDa) two times (one night and 3 h) against the same buffer as the one described above (except that the glycerol was not deuterated) but in 100% D2 O instead of 100% H2 O. The sample was then concentrated to 23 ␮M (Amicon threshold cut of 10 kDa). In the following, the buffers in 90% H2 O/10% D2 O and in 100% D2 O will be referred to as the H2 O and D2 O buffer, respectively. The main difference between the H2 O and the D2 O sample is that protons i and b are resolved in the H2 O buffer whereas they overlap in the D2 O buffer. When i and b overlap they are described by protons ib in the text. Protons e and j and protons h and g overlap in both buffers and are referred respectively to as protons ej and hg in the text. NMR experiments were performed at 298 K on a Bruker AVANCE II 500 MHz equipped with a xyz-gradient 5 mm BBI probe or on a Bruker AVANCE III 600 MHz equipped with a z-gradient QCI-HFCN Cryoprobe. All NMR experiments were recorded twice on a sample in H2 O buffer and a sample in D2 O buffer. The spectra were calibrated using the HEPES signal at 3.8 ppm. The reference 1D spectrum was recorded using an excitation sculpting scheme [17] with 64 scans, a relaxation delay of 4 s and an acquisition time of 1.9 s. This spectrum was used to evaluate the quality of the sample and to determine the concentration of GF109203X using an external reference sample of 8.1 mM maleic acid in a 3 mm tube and the Eretic/Pulcon

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principle [18,19]. STD/GEM-STD experiments [20] were recorded with 2048 scans using a relaxation delay of 4 s, an acquisition time of 1.9 s and saturation times of 0.5,1,2,3 and 4 s. The protein resonances were saturated using a 50 ms Eburp2 pulse applied at 300 Hz in a continuous fashion and using a peak radio-frequency power of 60 Hz leading to a saturation bandwidth of about 200 Hz. The off-resonance pulse of the reference experiment was applied at a frequency of 50,000 Hz. WaterLOGSY experiments [21] were recorded with 1024 scans using a 5 ms Gaussian pulse to selectively invert the water magnetization. DIRECTION experiments [22] were recorded with 64 scans and a relaxation delay of 10 s and an acquisition time of 1.9 s. The protein resonances during the variable relaxation times were saturated using the same 50 ms Eburp2 pulse as the one used in the STD experiment. The variable relaxation delay used to monitor the recovery of the 1 H magnetization was varied from 50 ms to 3 s. 2D tr-NOESY [23] and tr-ROESY [24] experiments were recorded with 512 increments in t1 and 4096 data points in t2 . The spectral width was 14 ppm in both dimensions. Sixty-four scans were recorded per t1 increment and water suppression was achieved using an excitation sculpting scheme. For tr-NOESY experiments, a gradient pulse was applied at the end of the mixing time to remove residual transverse magnetization and thus to improve the quality of the spectra. 2D tr-NOESY experiments were recorded with the following mixing times: 200, 400 and 600 ms. These experiments were recorded with and without water suppression during the mixing time in order to decrease the effect of spin diffusion relayed through water molecules (MINSY experiment [25]). 2D tr-ROESY experiments were recorded with a 300 ms mixing time. 2.3. Molecular docking Docking studies on Chk1 were performed on the available XRay crystallographic structure 1NVQ, solved at 2 A˚ resolution [26]. Chk1 kinase is co-crystallized (in an open conformation) with UCN-01 ligand in its ATP-binding pocket. Hydrogen atoms were added, amino acids were correctly charged at pH 7 and water molecules were removed. The added hydrogen atoms were minimized with the CHARMm force-field [27,28] from DiscoveryStudio [29] using steepest descent algorithm followed by conjugate gradient method. GF109203X molecule was ionized with Ionize from Chemaxon and converted to 3D with Corina. Docking, without constraint, was performed in the ATP-binding site of Chk1 with the program GOLD from CCDC (Cambridge Crystallographic Data Center) [30] using a Genetic Algorithm (GA) with the default settings i.e. # operations = 100,000. The molecule was docked within a radius of 14 A˚ around the mass center of UCN-01. 50 docking solutions were kept and we considered the top 10 best poses of GF109203X based on the default fitness function of GOLD. All these 10 poses have a fitness function better than 58. Structural overlay showed that these docking poses are closed to the crystallographic pose of UCN-01. 3. Results 3.1. Identification and characterization of Chk1 inhibitors by NMR Nineteen different chemical compounds and known Chk1 inhibitors [16,31] were screened against Chk1-cat-HT (1–289 amino acids) by NMR using STD and WaterLOGSY experiments in order to detect an interaction between them and the protein Chk1. The structure of all ligands was first checked with a 1 H NMR spectrum in DMSO-d6 . Then, the solubility of the ligand was checked by addition of 10 ␮l of the ligand solution into the protein buffer using again a 1 H NMR spectrum. The resulting tube contained roughly

3

Table 1 List of the 7 ligands studied by STD and WaterLOGSY experiments (nd: not determined).

S 40653-1 S 41479-1 S 38823-1 S 27788-1 S 41842-1 S 41842-2 GF109203X

Interaction by STD

Interaction by WaterLOGSY

Other

Yes Yes nd No Yes Yes Yes

Weak No Yes No Yes Yes Yes

Impure product Unstable product

3% DMSO-d6. Finally, a spectrum of the ligand with the protein was recorded. 89% of the ligands were soluble in DMSO-d6 at a concentration of about 10 mM and 63% of the ligands were not soluble in the protein buffer at a concentration of 250 or 500 ␮M. If the ligands were soluble in the buffer, they were also soluble with the protein. After this initial selection step, 7 ligands were found to be soluble with the protein and were therefore selected for the NMR study. STD and/or WaterLOGSY experiments revealed that 6 of them interacted with Chk1. The results are summarized in Table 1 and the chemical structure of the most potent compounds is shown in Fig. 1. Because of the presence of impurities in S 40653-1 and the instability of S 38823-1, our study focused on GF109203X (a staurosporine derivative), S 41479-1 and S 41842-2 (hymenialdisine derivatives) and S27788-1 (negative control). S 41842-1 was not studied because it is the same molecule as S 41842-2 but without the trifluoroacetic salt.

3.2. Interaction of GF109203X, S 41479-1 and S 41842-2 with Chk1 A solution containing 20 ␮M of Chk1 in the presence of 487 ␮M of GF109203X, 416 ␮M of S 41479-1 and 533 ␮M of S 41842-2 was then studied by 1 H NMR. The results of the STD (Fig. 2) and WaterLOGSY experiments (data not shown) prove that the three ligands bind to Chk1 since several signals belonging to these different ligands are observed in the aromatic part of the spectra. In the following, the analysis of the STD data will be performed using the STD factor defined as ASTD = (I0 − Isat )/I0 = ISTD /I0 where I0 is the intensity of the signal in the reference experiment and Isat is the intensity of the same signal in the saturated spectrum [20]. In order to determine the specificity of the interaction between GF109203X, S 41479-1, S 41842-2 and Chk1, the dissociation constant KD of these ligands was first measured by NMR. Several STD NMR experiments were performed with a constant concentration of Chk1 of 20 ␮M and the following concentrations for each of the three ligands: 100 ␮M, 250 ␮M, 500 ␮M and 950 ␮M. The resulting dissociation constants KD of GF109203X, S 41479-1 and S 41842-2 with Chk1 were found to be 204 ± 50 ␮M, 787 ± 2 ␮M and 408 ± 41 ␮M respectively (data not shown). These dissociation constants were determined exclusively by NMR using well resolved aromatic signals in order to obtain a better accuracy. They were then compared with the only available data in the literature, the IC50 values obtained with the flash plate assay method: GF109203X (IC50 = 348 nM), S 41479-1 (IC50 = 1.7 ␮M) [31] and S 41842-2 (IC50 = 19 nM) [16]. Even though the IC50 value is not a direct indicator of affinity, the KD and the IC50 value are in agreement for the three compounds studied. The relative affinity of the ligands with the Chk1 target was then studied using competition experiments by STD NMR. In order to check that the three ligands studied bind to the same site of Chk1, a STD experiment was recorded with a saturation time of 2 s for each of the following three samples: (1) a sample containing only

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a

2

b

S 40653-1

S 41479-1

S 38823-1

a

S 27788-1

S 41842-1

g

h

b

S 41842-2

c d

i f

a e

j k

l m n

GF109203X Fig. 1. Chemical structures of the most potent compounds tested by NMR (cf. Table 1).

Chk1 and GF109203X, (2) the same sample to which S41479-1 was added, (3) the previous sample to which S41842-2 was also added. The relative degrees of saturation obtained for the individual protons ASTD are presented in Fig. 3 for the three different samples studied. These results show clearly that each time a competing ligand is added to the initial solution of GF109203X with Chk1 (first addition of S 41479-1 and then addition of S 41842-2), the ASTD of the ring protons of GF109203X decreases. These data are clear evidence that the three ligands are all competing for the same site of Chk1. 3.3. Interaction of GF109203X with Chk1 Since GF109203X is the ligand that interacts with the highest affinity with Chk1 (KD = 204 ± 50 ␮M), we decided to focus our interest on this compound for the remainder of the study. The 1 H resonance assignment of GF109203X free in solution in the H2 O buffer without Chk1 was first established using standard twodimensional 1 H–1 H TOCSY and NOESY/ROESY experiments. The 13 C resonance assignment was established using two-dimensional gradient 1 H–13 C HSQC experiment. The 1 H/13 C chemical shifts and the intramolecular NOEs observed for the free form of GF109203X in the H2 O buffer are presented in Table 2 and Fig. 4, respectively.

Table 2 1 H and 13 C chemical shifts of GF109203X in solution without Chk1 in the H2 O buffer at 298 K. Assignment of GF109203X

1

H chemical shift (ppm)

13

f a e j d i b c hg k l m n

7.84 7.60 7.49 7.47 7.18 7.07 7.06 6.84 6.66 4.28 2.16 2.92 2.73

131.2 133.2 112.0 114.0 124.6 124.2 123.2 122.2 122.4 44.7 26.7 51.3 44.7

C chemical shift (ppm)

All the experiments used thereafter to study the interaction of GF109203X with Chk1 were performed on a 3 mm NMR tube containing 20 ␮M of Chk1 and 469 ␮M of GF109203X (molar ratio 1:24) either in the H2 O or the D2 O buffer. First, it is important to note that the 1 H chemical shifts of GF109203X in this sample

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a A f

a

hg

a ej d

8.4

8.3

8.2

8.1

8.0

7.9

7.8

7.7

7.6

7.5

7.4

7.3

b i

b

c

7.2

7.1

7.0

6.9

6.8

6.7

6.6

6.5

7.2

7.1

7.0

6.9

6.8

6.7

6.6

6.5

Chemical Shift (ppm)

B

8.4

8.3

8.2

8.1

8.0

7.9

7.8

7.7

7.6

7.5

7.4

7.3

Chemical Shift (ppm) Fig. 2. 1D 1 H NMR spectra of Chk1 at a concentration of 20 ␮M in the presence of 487 ␮M of GF109203X (purple stars), 416 ␮M of S 41479-1(blue stars) and 533 ␮M of S 41842-2 (pink star) acquired on a 600 MHz spectrometer with a cryoprobe at 298 K. Only the aromatic part of the ligands is shown on the figure. (A) Reference 1D excitation sculpting spectrum, (B) STD NMR spectrum recorded with a 2 s saturation time. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

b

g

h i

f

c d

a e

j k

l m n

Fig. 3. The relative degrees of saturation of the individual protons of GF109203X in interaction with Chk1 only (blue), after addition of S 41479-1 (red) and after addition of S41479-1 and S 41842-2 (green). The concentration of Chk1 was 20 ␮M and that of GF109203X, S41479-1 and S 41842-2 about 487 ␮M, 416 ␮M and 533 ␮M, respectively. The diagram is represented in such a way that the x-axis represents the position of the peaks in the spectrum shown in Fig. 2 (only the aromatic part). STD NMR spectra were recorded with a 2 s saturation time. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

are identical to those obtained in the sample without Chk1. This observation is in agreement with the fact that GF109203X is in fast exchange between the free form and the bound form. The 1 H longitudinal relaxation times of GF109203X in fast exchange with Chk1 were determined at 298 K (Table 3). The aliphatic protons of GF109203X have a longitudinal relaxation time shorter than 1 s whereas the aromatic protons of GF109203X have a longitudinal relaxation time comprised between 1.3 s and 1.9 s. The STD and WaterLOGSY experiments recorded in the H2 O buffer confirm that GF109203X interacts with Chk1 (Fig. 5). This interaction can be seen

NOE

Fig. 4. Chemical structure of the GF109203X ligand. The NOEs observed in the free state without the protein Chk1 and used for the assignment are indicated.

Table 3 1 H longitudinal relaxation times chemical shifts of GF109203X in solution with Chk1 in the H2 O buffer at 298 K (nd: not determined). Assignment of GF109203X

Longitudinal relaxation time T1(s)

f a ej d i b c hg k l m n

1.93 1.46 1.35 1.31 1.36 1.69 1.31 1.46 nd 0.71 nd nd

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A

f

8.5

8.0

a ej

7.5

d ib c hg 7.0

6.5

6.0

5.5

5.0

B

f

8.5

8.0

a ej

7.5

d ib c

7.0

4.5 4.0 3.5 Chemical Shift (ppm)

3.0

2.0

1.5

1.0

0.5

0

n

hg

6.5

2.5

6.0

5.5

5.0

4.5 4.0 3.5 Chemical Shift (ppm)

3.0

2.5

2.0

1.5

1.0

0.5

0

6.0

5.5

5.0

4.5 4.0 3.5 Chemical Shift (ppm)

3.0

2.5

2.0

1.5

1.0

0.5

0

C

f 8.5

8.0

a ej 7.5

d ib c hg 7.0

6.5

Fig. 5. 1D 1 H NMR spectra of Chk1 at a concentration of 20 ␮M in the presence of 469 ␮M of GF109203X (ratio 1:24) performed on a 500 MHz spectrometer at 298 K. (A) 1D 1 H reference spectrum (B) WaterLOGSY spectrum (1k scan, experiment time 2 h) (C) STD NMR spectrum (1k scan, experiment time 2.5 h, saturation time 2 s). STD and WaterLOGSY NMR spectra show that GF109203X binds to the protein Chk1. From the STD spectrum, one can characterize the binding epitope using the relative integral intensities of the signals in spectrum B.

on the aromatic (STD and WaterLOGSY experiments) and methyl protons (only in the WaterLOGSY experiment). 3.4. Group epitope mapping of the bound GF109203X with Chk1 by STD In order to characterize which part of GF109203X interacts the most with the target, group epitope mapping experiments were undertaken. These experiments are based on the fact that the ligand protons which are in closest interaction with the protein active site exhibit the highest STD factor [32,33]. The STD spectrum of Fig. 5C shows this effect since some protons clearly show a stronger STD response than others. To characterize more precisely this effect, a build-up curve was established using STD factors recorded at several saturation times (0.5 s, 1 s, 2 s, 3 s and 4 s). The whole series of experiments were performed in both H2 O and D2 O buffers. The results of these two sets of experiments are presented in Fig. 6A (H2 O buffer) and B (D2 O buffer). The T1 relaxation time of each proton is also indicated for both buffers. In the D2 O buffer, protons ib, ej and hg could not be resolved and it was therefore not possible to measure the individual STD factors. The same was also observed for protons ej and hg in the H2 O buffer. The main information that can be obtained from these figures is that the build-up curves of the STD factor in H2 O and D2 O show the same pattern for most protons. However, the intensity of the STD effect observed is clearly stronger in D2 O than in H2 O (an average ratio ranging from 3 to 4). This effect is well known and can be attributed to an effect of dilution of the STD effect in the H2 O buffer through rapidly exchanging water molecules between the ligand–receptor interface and the buffer solution [34,35]. One of the main differences observed between the two buffers is the fact that the proton f shows a much stronger STD intensity in D2 O than in H2 O. This is most likely

due to the presence of an exchangeable NH proton in alpha of proton f in GF109203X. Moreover its longitudinal relaxation time is much longer in D2 O (3 s) than in H2 O (1.9 s). The intensity of the STD effect of the different protons of GF109203X in H2 O and D2 O obtained with a saturation time of Chk1 of 1 s is represented directly on its chemical structure in Fig. 7A and B, respectively. In order to compare the results for the two different samples, the value of the largest STD factor in both cases was set to 100%. The ring protons c, d, ib and hg have all strong STD factors ranging from 69% to 100% in H2 O and from 86% to 100% in D2 O. On the other hand, protons ej and a exhibit smaller STD factors, ranging from 31% to 42% in H2 O and from 44% to 55% in D2 O. As noticed previously, one exception is proton f which has a STD factor of 41% in H2 O and of 80% in D2 O. The smallest value of the STD factor is found for proton l that reaches a value of only 38% in H2 O and 34% in D2 O. One must exert some caution when comparing the STD factors obtained on aromatic and aliphatic protons since it is known that relaxation times have a strong influence on the STD factor measured [32]. This is particularly true for proton l which has a much shorter relaxation time (T1 = 0.7 s) than the aromatic protons (T1 = 1.3–1.9 s). For this proton, the STD factor was also measured at a saturation time of 0.5 s and was found to be similar to the one obtained at 1 s. Overall, these STD data show that protons c, d, ib and hg interact the most with Chk1 and are buried within the active site of Chk1. The two indole rings of GF109203X show a slightly different behavior with the upper part of the ring containing protons b, c, d interacting slightly more with the target than the upper part of the ring containing the protons g, h, i. On the contrary, the data obtained for protons ej and a show clearly that the lower part of both indole rings does not interact as much with the target. Similarly, the STD intensity measured for the proton l of the aliphatic chain (at a saturation time of 0.5 s) tends to prove that this proton

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f T1=1.93s

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Fig. 6. Plot of STD factor of GF109203X bound to Chk1 against the saturation time (A) in H2 O buffer (B) in D2 O buffer. STD experiments were performed at 298 K on a 600 MHz spectrometer with a Cryoprobe. The 1 H T1 longitudinal relaxation times of each signal measured with a saturation of Chk1 during the variable relaxation delays are also presented.

A O 73%

H N

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85%

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41% 42%!

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31%

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87%

100%

86%

88%

80% 55%

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44%

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55%

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34%

Fig. 7. Structure of GF109203X displaying the relative degrees of saturation of the individual protons normalized to that of the c protons (A) in H2 O buffer (B) in D2 O buffer. The concentration of Chk1 was 20 ␮M and that of GF109203X 469 ␮M. The saturation time used for the STD experiment was 1 s.

does not interact as much with the active site either. For the other aliphatic protons (k, m and n), it was not possible to measure the STD factor because of either a superimposition of the signals with the buffer signals (protons m and n) or of baseline distortions due to the residual water signal (proton k). On an experimental note, it is worth mentioning that a STD factor can be obtained for the overlapping protons ib and ej using a 2D STD-TOCSY experiment [20]. However, in our case, the STD factors obtained with this experiment were very similar for protons i and b and for protons e and j and therefore did not provide any additional information (data not shown).

((T1irr − T1non irr )/T1irr ) was plotted for each proton of GF109203X. The experiments were recorded, as before, in the H2 O and in the D2 O buffer. The results of the DIRECTION experiment presented in Fig. 8 show that protons c, d, ib and hg interact the most with Chk1 and therefore confirm the STD results. Similarly, the DIRECTION experiment also indicates that protons ej and a do not interact as much with the target.

3.5. Group epitope mapping of the bound GF109203X with Chk1 by DIRECTION

The tr-NOESY experiment is based on the well-known fact that small ligands in solution exhibit NOEs that are vanishingly small and positive whereas bound ligands exhibit NOEs that are strong and negative [36,37]. The intensity of the observed cross-peaks in the resulting tr-NOESY experiment is the population weighted sum of the cross-relaxation rates in the free and the bound states. The trNOESY experiment will only provide valuable information on the bound conformation of the ligand if the fast exchange condition is fulfilled (KD > 10−6 M) [23], which is the case for GF109203X (measured KD value of 204 ␮M). Tr-NOESY experiments were recorded under identical conditions in the H2 O and the D2 O buffer. The tr-NOESY response is much stronger in D2 O than in H2 O which is a direct consequence of the dilution of the NOE effect by the H2 O molecules present at the ligand–receptor interface [34,35]. The presence of an exchangeable NH proton on GF109203X will also contribute in the H2 O buffer to the dilution of the NOE effect through an exchange of this proton with the free water molecules. The results of the 600 ms tr-NOESY experiment recorded in the D2 O buffer are shown in Fig. 9. For both samples, the results show the

Recently a new method for GEM called DIRECTION has been proposed [22]. This method utilizes the difference in the longitudinal relaxation times of the protons of the ligand recorded with and without irradiation of the protons of the target. This experiment is conceptually a T1 relaxation experiment recorded with and without selective saturation of the target protons during the relaxation period. The difference in the 1 H recovery rate generally corresponds to the intermolecular cross-relaxation term between the protons of the ligand and the receptor. It therefore reports on the proximity of the ligand protons with the protein surface and allows the identification of the part of the ligand that interacts with the target. This methodology was applied to GF109203X bound with Chk1. For both inversion-recovery experiments (with and without irradiation of the protons of the protein Chk1) the apparent longitudinal relaxation time T1irr and T1non irr were determined with the TOPSPIN 2.1 software and the percentage of variation in the T1 value

3.6. Structural information on the bound state of GF109203X by tr-NOESY and tr-ROESY

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Fig. 8. DIRECTION and STD-based epitope mapping of GF109203X bound to Chk1 (A) in H2 O buffer (B) in D2 O buffer. The STD factor (obtained with 1 s of saturation time) normalized to 100% is shown for each proton.

Fig. 9. 2D tr-NOESY spectrum of Chk1 at a concentration of 20 ␮M in the presence of 487 ␮M of GF109203X in D2 O recorded at 600 MHz with a Cryoprobe and 600 ms of mixing time. The most important transferred NOEs observed in this experiment and not present in the free form without Chk1 are indicated on the chemical structure of GF109203X.

expected NOE contacts within the aromatic protons and between some aromatic protons and the aliphatic chain. In particular, NOEs are detected between protons a and protons k, m, n and l of the aliphatic chain. NOEs are also detected between protons e and the aliphatic protons k and l. More surprisingly NOE cross-peaks are also visible between the h/g protons and the protons k, m, n and l of the aliphatic chain. The strongest of these cross-peaks corresponds to an interaction between h/g and the methyl protons of the aliphatic chain. Although some of these cross-peaks might be due to spin diffusion, they are clearly visible in the 600 ms NOESY experiment and they are also detected in the 200 and 400 ms

NOESY experiments. This observation was also confirmed by the results of a 300 ms tr-ROESY experiment recorded in the two different solvents. In particular, the cross-peak between h/g and the methyl groups are clearly identified in the tr-ROESY recorded in the D2 O buffer and are definitely missing in the spectrum recorded on GF109203X in the buffer solution without Chk1 (data not shown). Strong NOEs are also observed between protons a and h/g. Of interest, in the fact the no NOE correlation is detected between protons f and a, which proves that the two aromatic groups are not in the same plane. Overall, proton f shows only extremely weak correlations. The lack of NOEs of proton f could also be attributed to the

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4. Discussion 4.1. Comparison between the use of H2 O or D2 O buffer for NMR studies

Fig. 10. 3D model of GF109203X in interaction with Chk1 obtained by a combination of molecular modeling and NMR data.

presence of the exchangeable NH proton that reduces the intensity of the NOE through chemical exchange with the water molecules. Another interesting piece of information that can be obtained from tr-NOESY experiments is the degree of exposure of the protons of the ligand to the water molecules present in the sample. In the H2 O buffer, some of the aromatic protons show strong NOE cross-peaks with the water signal. More specifically, these protons are f, j, i and h/g which are all located on the same aromatic ring of GF109203X. Similar cross-peaks were also observed in tr-ROESY experiments. This observation tends to prove that this part of the molecule is a bit more exposed to the solvent and that some water molecules might be present at the ligand–receptor interface. These water molecules can have a very short residence time and are not always observed in the X-ray structure of ligands bound to their receptor. This observation emphasizes the point that NMR and X-ray crystallography are complementary techniques. 3.7. Molecular modeling of Chk1 All the 10 poses found by molecular modeling showed two hydrogen bonds between the maleimide carbonyl and NH of GF109203X and Cys87-NH and Glu85-CO of Chk1 backbone, respectively. These two interactions with the hinge region are usual for kinases [38]. These 10 poses can be clustered into 3 different docking solutions. Among the 3 different docking solutions of GF109203X, 2 of them showed interaction between the tertiary amine group and the acid side chain of Glu91 as the UCN-01 in its X-Ray structure or with carbonyl oxygen of Glu134 which is also closed. The second indole group can adopt two different orientations due to the rotation around the bond between indole and maleimide group. Both indoles made many favorable van der Waals interactions with the backbone and side chains of residues from the N-terminal domain including Tyr86, Leu15, Tyr20, Val23 and from the C-terminal like Asp148, Leu137 and Val68. Using the NMR results of the tr-NOESY and tr-ROESY experiment, it was possible to determine more precisely the position of GF109203X in the pocket of Chk1. Several positions were available and finally only one docking position showed an interaction compatible with the NMR results (Fig. 10). The protons h and g are near the lateral aliphatic chain of GF109203X and especially near the methyl protons n (trNOESY results) and the proton f is far from the proton a (tr-NOESY results). Moreover in this model, protons b, c, d seem to be slightly more buried in the pocket of Chk1 than protons gh, i which is in agreement with the results of the epitope mapping by STD and DIRECTION.

Proteins are typically available in a H2 O buffer which is not the most appropriate buffer for NMR studies due to a dilution of the STD and NOE effects by the H2 O molecules. We have shown in this study that the signal to noise ratio, for the same protein Chk1, is about 3 times better in D2 O than in H2 O for experiments like STD, tr-NOESY and tr-ROESY. It is therefore important to work in D2 O for this type of experiments where the detection of low intensity peaks is crucial. However, obtaining the protein in a deuterated buffer is not straightforward. A method like lyophilization is quite aggressive and might damage the protein. This method was clearly not adapted to Chk1 since no binding was detected with GF109203X after the lyophilization of the protein. An alternative method, which is more advisable, is to dialyse the protein against a D2 O buffer. This method was applied to Chk1 and was able to preserve the functions of the protein. The only drawback of the method is that the residual H2 O is more important than in samples obtained through lyophilization. It is important to note that for all these experiments (1D and 2D) the quality of the baseline is extremely important during the data analysis step and the quality of the data obtained is generally better in D2 O than in H2 O. It should be pointed out that working in a H2 O buffer offers some specific advantages such as being able to detect the effect of water molecules present at the ligand–receptor interface.

4.2. Comparison between STD and DIRECTION experiments The main difference between STD and DIRECTION experiments is that STD is a screening technique whereas DIRECTION is not. Indeed, STD experiments are able to screen batches of several ligands and extract those that interact with the target. DIRECTION experiments are not able to filter selectively the interacting ligands since no subtraction takes place in this experiment and the signals of all the ligand are simultaneously detected. For GEM experiments, DIRECTION definitely has some advantages over STD. First it can be significantly faster because of its higher sensitivity. This is due to the fact that 100% of the signal is detected in DIRECTION experiments whereas only a fraction of the signal is detected in the STD spectrum (i.e. the difference between the saturated and the nonsaturated spectrum). Secondly, DIRECTION experiments are less sensitive to differences in T1 values than STD experiments since only the relative variation in T1 is detected for each proton. It is therefore more adapted to study systems with a wide range of T1 values (aliphatic vs. aromatic protons). On the analysis side, data are analyzed faster with the DIRECTION method than with the STD method since only a standard fitting of the T1 relaxation data is necessary to obtain T1irr and T1non irr . For the STD method, the intensity or the integral of each signal in the reference and in the saturated spectra have to be measured, then the ratio (I0 − Isat )/I0 = ISTD /I0 must be calculated. It should be noted that both experiments are sensitive to an aggregation and a precipitation of the sample. The experiments should preferentially be performed on a fresh sample with no aggregation. We observed on most of our samples that some precipitation of the ligand at the bottom of the NMR tube occurred typically after 15 days at 298 K. A consequence of this precipitation is that the concentration of the ligand decreased with time. For example, a solution of GF109203X containing 20 ␮M of Chk1 and initially prepared with a concentration of 410 ␮M, was found to contain only 250 ␮M of ligand in solution after two weeks.

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4.3. Structure of the ligand GF109203X in the bound state to Chk1 We have shown in this work that NMR experiments could provide valuable structural information on the structure of the GF109203X ligand bound to the protein Chk1. The tr-NOESY and tr-ROESY experiments provided crucial data that, when use in conjunction with molecular modeling, allowed us to determine the structure of GF109203X in the binding pocket of Chk1. The resulting molecular model presented in Fig. 10 fulfills most of the NOE and ROE contacts that were detected in tr-NOESY and tr-ROESY experiments. This model also shows that protons b, c and d are slightly more buried in the cavity of Chk1 than protons h, g and i which is in agreement with the STD and DIRECTION results. The conformation of GF109203X presented in Fig. 10 is clearly only adopted in the bound state since the NOEs observed between the protons hg and the methyl protons are only seen in the presence of the protein Chk1 and not in the buffer solution alone. 5. Conclusion In this paper, we have used NMR based screening techniques (STD and WaterLOGSY) to identify small therapeutic molecules that interact with the DNA damage checkpoint enzyme Chk1. Using these experiments, it was possible to identify 6 ligands that interacted with the protein. STD competition binding experiments enabled us to prove that GF109203X, S 41479-1 and S 418422 compete for the same binding site. STD experiments further allowed us to determine the constant dissociation of these three ligands. GF109203X was found to be the most potent inhibitor and its mode of interaction with Chk1 was studied in more detail. The use of STD and DIRECTION experiments allowed us to determine the binding epitope of GF109203X whereas tr-NOESY and tr-ROESY experiments allowed us to obtain structural information on the bound conformation of GF109203X to Chk1. Using these NMR results and docking protocols, a 3D model of the complex between GF109203X and Chk1 was proposed. These results show that NMR based screening techniques, although not as sensitive as other commonly used methods such as fluorescence, radiolabeling, surface plasmon resonance or mass spectrometry [39], are able to supply a wealth of information. The information obtained by NMR is clearly complementary to the information obtained by X-ray in the solid state. As a last and important point, we have shown that these techniques can be used efficiently on fairly small quantities of protein (155 ␮g corresponding to 4.6 nmol) and ligand (43 ␮g/104 nmol) using 3 mm NMR tubes, modern NMR instrumentation and optimized experimental conditions. Acknowledgements The authors thank Benjamin Fould and Anne-Laure Pommier for the dialysis of the protein in the HEPES buffer in 100% D2 O.

[5]

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[12]

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[15] [16]

[17]

[18] [19]

[20]

[21]

[22]

[23] [24]

[25]

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Please cite this article in press as: N. Lancelot, et al., Applications of NMR screening techniques to the pharmaceutical target Checkpoint kinase 1, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.10.032