Custom chemical microarray production and affinity fingerprinting for the S1 pocket of factor VIIa

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 335 (2004) 50–57 www.elsevier.com/locate/yabio

Custom chemical microarray production and aYnity Wngerprinting for the S1 pocket of factor VIIa Stefan Dickopfa, Michael Franka, Hans-Dieter Junkera, Sabine Maiera, Günther Metza, Holger Ottlebena, Harald Raua,1, Nathalie Schellhaasa, Kristina Schmidta, Renate Sekula, Cecile Vaniera, Dirk Vettera,1, Jörg Czechb, Martin Lorenzb, Hans Matterb, Manfred Schudokb, Herman Schreuderb, David W. Willb, Hans Peter Nestlerb,¤ a

GraYnity Pharmaceuticals AG, Im Neuenheimer Feld 518-519, D-69120 Heidelberg, Germany b Aventis, Industrial Park Hoechst, Building G 879, D-65926 Frankfurt am Main, Germany Received 4 June 2004 Available online 8 October 2004

Abstract The goal of this study was to explore the applicability of surface plasmon resonance (SPR)-based fragment screening to identify compounds that bind to factor VIIa (FVIIa). Based on pharmacophore models virtual screening approaches, we selected fragments anticipated to have a reasonable chance of binding to the S1-binding pocket of FVIIa and immobilized these compounds on microarrays. In aYnity Wngerprinting experiments, a number of compounds were identiWed to be speciWcally interacting with FVIIa and shown to fall into four structural classes. The results demonstrate that the chemical microarray technology platform using SPR detection generates unique chemobiological information that is useful for de novo discovery and lead development and allows the detection of weak interactions with ligands of low molecular weight.  2004 Elsevier Inc. All rights reserved.

The S1 clan of serine proteases is one of the largest protein families and includes key members of many physiological and pathophysiological processes such as the coagulation enzymes around thrombin, factor Xa, and factor VIIa (FVIIa),2 as well as the kallikreins and tryptases, involved in inXammatory processes. Some of these proteases have been under investigation for drug discovery for a long time. Recently, a thrombin inhibitor and its prodrug form, Ximelagatran, have been successfully approved in phase 3 clinical trials for the prevention of *

Corresponding author. Fax: +49 69 305 942 804. E-mail address: [email protected] (H.P. Nestler). 1 Present address: Complex Biosystems, Im Neuenheimer Feld 584, D-69120 Heidelberg, Germany. 2 Abbreviations used: FVIIa, factor VIIa; NMR, nuclear magnetic resonance; SPR, surface plasmon resonance; TFA, triXuoroacetic acid; HPLC/MS, high-performance liquid chromatography/mass spectrometry; TF, tissue factor. 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.08.033

stroke in atrial Wbrillation [1]. The deWning structural feature of this class of proteases is a deep S1 pocket that accommodates a lysine or an arginine in their natural substrates and is commonly addressed with a benzamidine or guanidine moiety in the search for inhibitors [2]. Although very attractive for the development of inhibitors, this highly basic group causes signiWcant problems during the pharmacokinetic optimization of drug candidates. For one subgroup of S1 clan proteases with an alanine in position 190 (e.g., thrombin, factor Xa), neutral P1 haloaromatic groups were found to maintain binding aYnity, whereas no neutral replacements have been reported for the 190Ser representatives such as FVIIa. We have engaged in the search for inhibitors of FVIIa with therapeutic potential, facing the pharmacokinetic challenge imposed by the benzamidine [3,4]. In this study, we report a fragment-based approach in the high-throughput quest for benzamidine replacements for FVIIa inhibitors.

S. Dickopf et al. / Analytical Biochemistry 335 (2004) 50–57

Methods and results During recent years, there has been growing interest in aYnity-based screening approaches using small molecules that probe single pockets of target proteins instead of the entire binding site (i.e., fragments). Detection of the binding via nuclear magnetic resonance (NMR) or crystallography [5–8] has the drawback of very limited throughput. Fragment-based low-aYnity microarray screening technology [9–11] allows the rapid experimental characterization of protein-binding properties and the identiWcation of potential ligands. To this end, small organic molecules are immobilized via a ChemTag linker [12] on a gold-covered chip surface as a spatially addressable microarray and are incubated with the protein or proteins of interest. Binding of the protein to speciWc molecules on the array is detected and quantiWed by wavelength shifts of the surface plasmon resonance (SPR). This highly parallel, labelfree detection technology oVers major advantages over classical screening approaches: (i) low-aYnity protein– ligand interactions can be detected, (ii) no assay development is required, (iii) high reproducibility can be obtained, (iv) high-throughput identiWcation of protein– ligand interactions is feasible, and (v) previous knowledge of protein function is not essential. Because of these characteristics, we applied this technology for the search of novel fragments binding to FVIIa.

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The fragments in this study were selected from commercial sources (MDL Available Compounds Directory, MDL Information Systems, San Leandro, CA, USA) using a three-dimensional pharmacophore model of the S1 pocket of FVIIa derived from crystallographic information of FVIIa with inhibitors [13–15] as well as activity information of benzamidine-based inhibitors [3,4]. Before the docking into the pharmacophore model, several Wltering criteria were applied to reduce the number of structures to be docked to approximately 30,000. Molecular weight of fragments was restricted to a maximum of 350, the number of rotatable bonds was limited to four, and compounds containing reactive groups were excluded. After visual inspection of structures with the best Wt in the pharmacophore docking, 1412 fragments with four diVerent reactive groups were chosen, allowing six diVerent coupling chemistries to the ChemTag molecules (Fig. 1). Amines and anilines were coupled by four diVerent methods: (i) amide formation, (ii) reductive amination, (iii) nucleophilic substitution, and (iv) urea formation. Carboxylic acids were coupled to the amino functionalized ChemTag using standard active ester coupling methodology [5]. Aldehydes and ketones were coupled to a hydroxylamine-modiWed ChemTag as oximes [6]. For synthesis of the ChemTag-fragment conjugates, ChemTag molecules were coupled to commercially available solid phase synthesis material (Amino-PEG-

Fig. 1. Overview of coupling chemistries used.

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S. Dickopf et al. / Analytical Biochemistry 335 (2004) 50–57

Table 1 Overview of synthesis results Type of compound

Fragments attempted

Coupling chemistry (Fig. 1)

Fragments successfully coupled

Amines and anilines

1031

Carboxylic acids Aldehydes Ketones Total

208 66 107 1412

1 (Amide formation) 2 (Reductive amination) 3 (Nucleophilic substitution) 4 (Urea formation) 5 (Amide formation) 6 (Oxime formation) 6 (Oxime formation)

630 154 437 91 164 46 37 1559

PP-Membrane, AIMS ScientiWc Braunschweig [10]) and immobilized in 96-well microtiter plates. Subsequently, the selected fragments were linked to the resin-bound ChemTag using the six diVerent coupling chemistries outlined in Fig. 1, and the ChemTag-fragment conjugates (i.e., array compounds) were cleaved from the synthesis resin with triXuoroacetic acid (TFA). After evaporation, the compounds were dissolved in aqueous buVer, transferred to 384-well microtiter plates, and stored for quality control and array production. Quality control was performed by high-performance liquid chromatography/mass spectrometry (HPLC/MS). Table 1 summarizes the number of selected fragments in each class and the number of products that were obtained with a minimum purity of 75% and single impurities less than 20%. For array production, 1534 ChemTag-fragment conjugates were selected and transferred into 384-well microtiter plates. In two wells, para-aminobenzamidine was placed as a positive control because this compound is known to bind to the S1 site in FVIIa. Nine array copies, each containing 1536 spots, were produced. Aliquots of the solutions were transferred into 384-well microtiter plates and diluted with spotting buVer (17.5% ethylene glycol, 175 mM NaH2PO4, 8.75 mM EDTA, 6.25% acetonitrile) to a Wnal fragment concentration of 50 M. Transfer onto the gold surface (coated with a self-assembling monolayer of aliphatic monomercaptams that were partially modiWed with maleinamide to capture the ChemTag thiol) was performed using robotic equipment with metal pins. FVIIa binding to the immobilized array compounds was detected and quantiWed using wavelength-dependent measurement of the SPR eVect. The two-dimensional sensor array is imaged onto a spatially resolving detector, and the light illuminating the sensor array is measured scanning the spectrum. Thus, the resonance spectrum of each single-sensor Weld of the array is obtained. The shift in SPR wavelength is a measurement for protein binding to the ligands immobilized on the sensor array. Proteins were incubated on the microarray in screening buVer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 8.15, 0.005% Tween 20). SPR wavelength shifts were measured, and the data were transferred into

GraYnity’s proprietary JARRAY software for further interpretation. FVIIa and tissue factor (TF) were incubated on the microarrays in a molar ratio of 1:1.5 for 2 h prior to measurement of wavelength shifts of the SPR signals. Experiments were carried out in duplicate on two microarray copies. The binding pattern in both FVIIa/TF experiments was reproducible with maximum wavelength shifts of the SPR signals of 2.3 or 2.1 nm in duplicate. The two positive control (para-aminobenzamidine) spots generated shifts of 0.2 and 0.1 nm with FVIIa/TF and were among the top 60 hits. However, the absolute intensities obtained under these experimental conditions were too low to allow unambiguous detection of the positive controls. In a control experiment, TF and FVIIa were incubated separately with other microarray copies. TF alone showed no signiWcant binding, with a maximum shift of 0.38 nm. FVIIa alone showed a pattern similar to that in the FVIIa/TF experiment but with a lower dynamic range. Thus, the array compounds interact speciWcally with FVIIa, and no signiWcant diVerence in binding speciWcity of FVIIa with or without TF to the ligands was observed (Table 2). Because the SPR eVect depends on the mass of analyte bound to the surface, we anticipated that the addition of an
-FVIIa-speciWc antibody to the screening experiment would increase detection signals without aVecting background binding. Therefore, an
-FVIIa

Table 2 Summary of aYnity Wngerprinting experiments performed Study number

Experiment

Result

1

FVIIa + TF

2

FVIIa

3 4 5

TF FVIIa + TF + Antibody FVIIa + Antibody

6 7

TF + Antibody Antibody

Signal pattern as in study 4; response intensity between study 2 and study 4 Signal pattern as in study 4; weak response No binding detected Signals shown in Fig. 2A No enhancement compared with study 1; no extra signals No binding detected No binding detected

S. Dickopf et al. / Analytical Biochemistry 335 (2004) 50–57

antibody (Kordia Laboratory Supplies, lot AP-632AR2, catalog SAFVII-AP, sheep
-human FVIIa, aYnity-puriWed IgG) was applied to the arrays after incubation with FVIIa/TF. After 1 h, the SPR wavelength shifts were recorded (Fig. 2A). The experiments showed that the addition of antibody resulted in strongly increased signals, with maximal wavelength shifts of 6.3 nm and with good reproducibility for the duplicate experiments (Fig. 2B). TF and antibody without FVIIa resulted in negligible wavelength shifts of less than 1 nm. The increase in signal intensities in the antibody-enhanced aYnity is speciWc because background binding seems not to be enhanced. A correlation analysis of the experiments with and without antibody showed that signiWcant signals are enhanced only by a factor of approximately 3 after the addition of antibody (Fig. 2C). This indicates that the addition of antibody generates better signal-to-noise ratios, leading to a more accurate detection of protein–ligand interactions. The interaction strengths, as assessed by the recorded wavelength shifts, were ranked. A total of 60 ligands, including the positive control para-aminobenzamidine, showed reproducibly signiWcant binding intensities at more than 3 above background. Because the addition of antibody strongly increased signal intensities without

53

aVecting background, an improved signal-to-noise ratio was obtained. The para-aminobenzamidine controls were unequivocally identiWed as interacting with FVIIa. Because the Ki of para-aminobenzamidine against FVIIa is approximately 7 £ 10¡5 M [16], we estimate the approximate detection limit based on the detected wavelength shifts to be in the range of high micromolar aYnities in this study (Table 3). The identiWed binders fall into various structural classes, with the most prominent being dihalogen- or dinitro-phenols, an aromatic carboxylic acid in the ortho position to the ChemTag, and sulfonamides where the amino-terminal substitution is a heteroaromatic or biphenylic residue. Based on biochemical activities, 18 of these 60 fragments were initially selected for conWrmation by crystallography. To facilitate crystallization trials, we selected trypsin as a surrogate of FVIIa. However, because it turned out that binding tendencies in the crystal could not be predicted from Ki values (due to crystal packing and physicochemical reasons), we selected a total of 47 fragments for soaking with trypsin in a second round. Trypsin was crystallized in the presence of benzamidine to prevent autodegradation during the crystallization. During the Wrst soaks, it turned out that benzamidine

Fig. 2. Results of the antibody-enhanced FVIIa and TF Wngerprints. (A) Top 60 interacting compounds displayed on map. The arrows indicate positions of benzamidine positive controls. The color-coding range indicates wavelength shifts between 0.0 nm (blue) and 6.5 (yellow) in the experiment. (B) Correlation of duplicate experiments of the FVIIa/TF/antibody experiment. Numbers on axes indicate nanometer wavelength shifts. (C) Correlation of FVIIa/TF experiment with the experiment including antibody. Numbers on axes indicated nanometer wavelength shifts. (For interpretation of the references to color in this Wgure legend, the reader is referred to the web version of this article.)

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S. Dickopf et al. / Analytical Biochemistry 335 (2004) 50–57

Table 3 Structures of the 20 fragments with highest aYnity to FVIIa in the SPR screen structures, conjugation chemistry, wavelength shifts, and % inhibitory activity against FVIIa at 1 mM

Oxim (6), 6.494 nm, 10%

Amide (5), 4.207 nm, 13%

Amide (5), 5.931 nm, 0%

Amide (5), 4.190 nm, 12%

Oxim (6), 5.517 nm, 25%

Amide (5), 4.049 nm, ¡1%

Amide (5), 5.491 nm, 9%

Amide (5), 4.010 nm, ¡4%

Amide (5), 5.360 nm, ¡5%

Amide (5), 3.542 nm, 4%

Amide (5), 5.096 nm, 26%

Amine (3), 3.486 nm, 4%

Amide (5), 4.944 nm, 0%

Oxim (6), 3.382 nm, 94% at 0.5 mM

Amide (5), 4.811 nm, 12%

Amide (5), 3.273 nm, 7%

Amide (5), 4.733 nm, 5%

Amide (5), 3.256 nm, 12%

Amide (5), 4.411 nm, 6%

Amide (5), 3.233 nm, 6%

was still present in the P1 pocket after soaking. To replace benzamidine directly, we aimed to dissolve the fragments to 100 mM in artiWcial mother liquor, but 30

of the 47 fragments were poorly soluble, and this was one potential reason for the low success rate in soaking. Therefore, we modiWed the soaking protocol by

S. Dickopf et al. / Analytical Biochemistry 335 (2004) 50–57

transferring the crystals to artiWcial mother liquor twice without benzamidine but with the fragment present. After this improved soaking procedure, there was no longer benzamidine present in the active site. As some of the compounds were tested more than once, we collected a total of 61 trypsin data sets (18 diVerent ligands) from these 47 fragments (Table 4). Only 1 fragment was found to bind to the P1 pocket, and only 1 fragment was found to bind to a crystal contact.

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The X-ray structure obtained with a fragment was that of dichloro-aminophenol. (Fig. 3). This structure shows an intricate hydrogen bonding network of the amino group with the polar residues, 189Asp and 190Ser, at the bottom of the S1 pocket. This binding mode could also be conWrmed in the cocrystal structure in FVIIa, although these crystals provided only a limited resolution (3.1 Å). To our knowledge, this is the Wrst low-basicity ligand reported in the S1 pocket of FVIIa.

Table 4 Summary of crystallization trials, results are listed for the original and modiWed soaking conditions described in the text Structure

Outcome X-ray result P1 pocket

Structure

Outcome X-ray result P1 pocket

Benzamidine ! empty

Benzamidine ! empty

Benzamidine ! fragment in crystal contact

Benzamidine ! empty

Benzamidine ! empty

Benzamidine ! acyl-derivative of enzyme (from autolysis?)

Benzamidine ! not interpretable protein, peptide bound (autolysis?)

Benzamidine ! empty

Benzamidine ! empty

Benzamidine ! very little density

Benzamidine ! empty

Benzamidine ! fragment

Benzamidine ! empty

Benzamidine ! empty

Benzamidine ! empty

Benzamidine ! empty

Benzamidine ! empty

Benzamidine ! empty

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S. Dickopf et al. / Analytical Biochemistry 335 (2004) 50–57

platform established at GraYnity generates unique chemobiological information that is useful for de novo discovery and lead development and that allows the detection of weak interactions with ligands of low molecular weight. The incorporation of these fragments into larger FVIIa inhibitors, which also interact with other pockets in the enzyme, has been in progress and will be reported in due course.

References

Fig. 3. Hydrogen bonds of trypsin (1.8 Å) and dichloro-aminophenol. The NH2 group is pointing toward 19Asp at the bottom of the S1 pocket and is at hydrogen bonding distance of the carboxylate of 189 Asp, the side chain OH and the main chain O of 190Ser, and two bound water molecules. The hydroxyl group is involved in a hydrogen bond with the bound sulfate ion. One of the chlorine atoms is at close distance (2.7 and 3.2 Å) to the OH of 195Ser and one oxygen atom of the bound sulfate ion.

Summary The goal of this study was to explore the applicability of SPR-based fragment screening to identify compounds that bind to FVIIa. Based on pharmacophore models and virtual screening approaches, we selected fragments anticipated to have a reasonable chance of binding to the S1-binding pocket of FVIIa. Because these fragments had molecular weights of only approximately 200 Da, the major challenge was the detection of lowaYnity protein–fragment interactions. We conjugated 1536 fragments to ChemTag spacer molecules, immobilized these array compounds on microarrays, and performed SPR-based aYnity Wngerprinting experiments for FVIIa with TF. In aYnity Wngerprinting experiments, a number of compounds were identiWed to be speciWcally interacting with FVIIa and shown to fall into four structural classes. In follow-up experiments, we conWrmed the interaction of fragments with the active site of FVIIa by enzymological studies of their biochemical activity and structural investigation by crystallography. Although the detected aYnities did not translate directly into biochemical activities or tendency of forming cocrystals, the results demonstrate that the chemical microarray technology

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