Isolation of Potent and Specific Trypsin Inhibitors from a DNA

Luca Mannocci*, Samu Melkko, Fabian Buller, Ilona Molnàr, Jean-Paul Gapian Bianké, Christoph E. Dumelin, Jörg Scheuermann, and Dario Neri*. Institu...
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Bioconjugate Chem. 2010, 21, 1836–1841

Isolation of Potent and Specific Trypsin Inhibitors from a DNA-Encoded Chemical Library Luca Mannocci,*,†,§ Samu Melkko,†,§ Fabian Buller,‡ Ilona Molnàr,‡ Jean-Paul Gapian Bianké,‡ Christoph E. Dumelin,§ Jo¨rg Scheuermann,‡ and Dario Neri*,‡ Institute of Pharmaceutical Sciences, ETH Zu¨rich, and Philochem AG c/o ETH Zu¨rich, Wolfgang-Pauli-Strasse, 10, 8093 Zu¨rich, Switzerland. Received April 23, 2010; Revised Manuscript Received July 15, 2010

Collections of chemical compounds, individually attached to unique DNA fragments serving as amplifiable identification bar codes, are generally referred to as “DNA-encoded chemical libraries”. Such libraries can be used for the de novo isolation of binding molecules against target proteins of interest. Here, we describe the synthesis and use of a DNA-encoded library based on benzamidine analogues, which allowed the isolation of a trypsin inhibitor with an IC50 value of 3.0 nM, thus representing a >10 000-fold potency improvement compared to the parental compound. The novel trypsin inhibitor displayed an excellent selectivity toward other serine proteases. This study indicates that DNA-encoded libraries can be used for the facile “affinity maturation” of suboptimal binding compounds, thus facilitating drug development.

INTRODUCTION The isolation of potent enzyme inhibitors is a frequent challenge in medicinal chemistry. Ligands with high binding affinity and specificity to a target enzyme typically allow the administration of low drug doses and minimize the risk of nontarget-related side effects. The medicinal chemical optimization of lead compounds frequently improves drug potency by several orders of magnitude (1), but often requires the synthesis and screening of hundreds of synthetic derivatives. In order to facilitate the synthesis and screening of chemical libraries, the tagging of individual compounds with DNA fragments serving as amplifiable identification bar codes has been proposed (2-18). Indeed, both DNA-encoded chemical libraries displaying a single molecule on DNA (“single-pharmacophore” chemical libraries) (13-18) and libraries with a molecule at the extremity of duplex-forming complementary strands (“dual-pharmacophore” chemical libraries) (6-12) can be used. While the de novo isolation of binders from DNA-encoded chemical libraries represent a valuable tool for drug discovery, most frequently the pharmaceutical industry needs efficient methods for the improvement of lead compounds, a procedure often termed “affinity maturation”. Trypsin is a good model to test chemical strategies for the development of inhibitors, since the function of the enzyme is very well understood at the molecular level (19, 20) and proteases are a class of important pharmaceutical targets (21). Dual pharmacophore chemical libraries have previously been used for the improvement of benzamidinebased trypsin inhibitors (11). However, no affinity maturation procedures have so far been reported for single pharmacophore chemical libraries, which in principle should more rapidly yield molecules with the desired properties, as they do not require * Corresponding authors. Prof. Dr. D. Neri, Institute of Pharmaceutical Sciences, ETH Zu¨rich, Wolfgang-Pauli-Str, 10, 8093 Zu¨rich (Switzerland), Fax: +41-44-6331358. E-mail: [email protected]. Dr. Luca Mannocci, Philochem AG c/o ETH Zu¨rich, WolfgangPauli-Str, 10, 8093 Zu¨rich (Switzerland), E-mail: luca.mannocci@ philochem.ch. † Both authors contributed equally to the manuscript. ‡ Institute of Pharmaceutical Sciences. § Philochem AG.

the optimization of linkers, joining the two individual binding fragments (11). In this work, we describe the synthesis and use of a single pharmacophore DNA-encoded affinity maturation library based on benzamidine, from which we isolated a trypsin inhibitor with an IC50 value of 3.0 nM, thus representing a >10 000-fold potency improvement compared to benzamidine.

MATERIALS AND METHODS Synthesis of BAM8000 compounds: The 8000 members of the BAM8000 library were synthesized in two-step “split and pool” amide-forming reactions. Initially, 40 Fmoc-protected amino acids containing a benzamidine moiety were chemically coupled to 5′-amino-modified 45-mer oligonucleotides following activation of the carboxylic acid in 70% (v/v) DMSO/water, with N-hydroxysulfosuccinimide, N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide and subsequent addition of aqueous triethylamine hydrochloride solution buffer, pH 10.0. The oligonucleotides had general structures: 5′-GGA GCT TGT GAA TTC TGG XXX XXX XXX GGA CGT GTG TGA ATT GTC-3′, where XXX XXX XXX unambiguously identifies the individual Fmoc-protected amino acid compound. All coupling reactions were stirred overnight at 30 °C. Subsequently, the reactions were quenched and simultaneously Fmoc deprotected by addition of piperidine in DMSO. The reactions were then purified by HPLC, and the desired fractions were dried under reduced pressure, redissolved in water and analyzed by LCESI-MS. Typical coupling yields were >39% overall. 4.0 nmol aliquots of each DNA-compound conjugate were pooled to generate a 40 member DNA encoded sublibrary. In the following split and pool step, the oligonucleotide sublibrary pool was aliquoted in 200 reaction vessels and an amide-forming reaction with a distinct amino reactive building block was performed following a very similar procedure as reported above (see Supporting Information). The identity of the compound used for the coupling reactions was encoded by annealing unique 44-mer oligonucleotides with general structure 5′-GTA GTC GGA TCC GAC CAC XXXX XXXX GAC AAT TCA CAC ACG TCC-3′ (where XXXX XXXX unambiguously identifies the individual carboxylic acid compound), partially complementary to the first oligonucleotide carrying the chemical

10.1021/bc100198x  2010 American Chemical Society Published on Web 08/31/2010

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Scheme 1. Synthesis and Encoding of the BAM8000 Librarya

a Initially, 40 different Fmoc-protected amino acids, containing a benzamidine moiety, were coupled to unique oligonucleotide derivatives, carrying a primary amino group at the 5′ extremity and a 9 base pair coding region. Subsequently, the oligonucleotide conjugates were pooled and coupled to 200 amino reactive building blocks (carboxylic acid, isocyanate, sulfonyl chloride, or cyclic anhydrides) in parallel reactions. The identity of each building block was encoded by means of a Klenow polymerization step, using a set of partially complementary oligonucleotides. The procedure resulted in an 8000-member library (BAM8000), in which each chemical compound was covalently attached to a double-stranded DNA fragment, containing two coding domains which unambiguously identify the compound’s structure (i.e., the two chemical moieties used for compound synthesis).

modification, and subsequently incubated with Klenow polymerase at 37 °C for 1 h. After purification on chromatographic cartridges, the 200 purified reactions were dissolved in 100 µL of water each and pooled to generate the 8000 member library (BAM8000) to a final total oligonucleotide concentration of 300 nM. Details of the synthesis, analytics, and structures of the 40 Fmoc-protected amino acids as well as the structures of the 200 amino-reactive building blocks used can be found in the Supporting Information online. Selection and decoding experiments: 50 µL of the library BAM8000 (20 nM, total oligonucleotide concentration) was added either to 50 µL trypsin-sepharose slurry (coating density as detailed in the text) or to sepharose slurry without trypsin. Both resins were preincubated with PBS and 0.3 mg/mL herring sperm DNA. After incubation for 1 h at 25 °C, the beads were washed 4 times with 400 µL PBS (20 mM NaH2PO4, 30 mM Na2HPO4, 100 mM NaCl), and used as template for PCR amplification of the selected codes. The PCR primers had the following structure: DEL_P1_A (5′-GCC TCC CTC GCG CCA TCA GGG AGC TTG TGA ATT CTG G-3′) and DEL_P2_B (5′-GCC TTG CCA GCC CGC TCA GGT AGT CGG ATC CGA CCA C-3′). The primers additionally contain at one extremity a 19 bp domain (underlined) required for highthroughput sequencing with the 454 Genome Sequencer system. The PCR products were purified with chromatographic cartridges. Subsequent high-throughput sequencing was performed

on a 454 Life Sciences-Roche FLX Sequencer platform. Analyses of the codes from high-throughput sequencing were performed by an in-house program written in C++. The frequency of each code has been assigned to each individual oligonucleotide conjugate. Synthesis of the binding molecules: In a polypropylene syringe, either 65 mg (30 µmol) of H-Gln-2-Chlorotrityl resin (Novabiochem, cat. no 04-12-2806) or 100 mg (30 µmol) of H-Ala-2-Chlorotrityl resin (Novabiochem, cat. no 04-12-2802) was suspended in a mixture of the appropriate Fmoc-protected amino acid (60 µmol, 1 mL), HBTU (120 µmol, 1 mL), and N,N-diisopropylethylamine (240 µmol, 0.5 mL) in dry DMF. After overnight incubation at 25 °C, the resin was washed 6 times with 2 mL dry DMF, and the fmoc moiety was removed by addition of 1 mL piperidine (50% in dry DMF) for 1 h at 25 °C. After washing 6 times with 2 mL dry DMF, the corresponding compound (60 µmol, 1 mL DMF) was added, and a further amide bond formation reaction was performed either as described above (carboxylic acid compounds) or by adding DIEA (Fluka, 240 µmol, 0.5 mL) in dry DMF (sulfonyl chloride and isocyanate compounds) for 6 h at rt. The resulting product was cleaved from the resin by treating the resin 5 times with 2 mL TFA (2% in CH2Cl2). The methylene chloride fractions were washed 3 times with water, dried on Na2SO4, and concentrated in vacuo. The crude carboxylic acid product was suspended in DMF (200 µL) and a solution of N-hydroxysulfosuccinimide

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Mannocci et al.

Figure 1. Plots representing the frequency (i.e., sequence counts) of the 8000 library members after selection on 4 different trypsin resins, as revealed by high-throughput 454 sequencing (19). The four panels are ordered from top to bottom according to decreasing protein coating density (a,b,c,d). The chemical structures of some of the most relevant trypsin binders are indicated, together with the identification number.

(36 µmol, 333 mM DMF/water, 2:1 v/v) and N-ethyl-N′-(3dimethylaminopropyl)-carbodiimide hydrochloride (33 µmol, 100 mM DMF) was added. After 1 h at rt, 4-(aminomethyl)benzimidamide dihydrochloride (30 µmol, ABCR, cat. no AB169853) and DIEA (180 µmol) were added. Following overnight incubation at 25 °C, HPLC purification was performed on a Synergy Prep RP18 column (5 µm, 10 × 150 mm) using a linear gradient from 20% to 100% MeCN 0.1% TFA, the desired fractions were collected and lyophilized. ESI-MS analysis confirmed the expected mass of the products. Details of the analytics can be found in the Supporting Information online. Trypsin inhibition assay: Bovine pancreatic trypsin (1 nM) in PBS (20 mM NaH2PO4, 30 mM Na2HPO4, 100 mM NaCl [pH 7.4], containing 10% DMSO) was incubated with different concentrations of inhibitors in 384-well microtiter plates (Greiner) for 30 min at room temperature. The reaction was started by the addition of the fluorogenic substrate Z-GGR-AMC (Bachem, Bubendorf, Switzerland) dissolved in PBS and 10% DMSO to an end concentration of 0.1 mM in a total volume of 100 µL. The change of fluorescence signal (ex: 383 nm; em: 455 nm; cutoff filter: 420 nm) was recorded over 10 min using a SpectraMax microplate reader (Molecular Devices). The rate of fluorescence signal increase over time (reaction velocity V) was plotted against the corresponding inhibitor concentrations, and the IC50 value for the inhibitor was obtained by fitting with equation V ) a × 0.5 × {-([Io] - [Eo] + IC50) + [([Io] [Eo] + IC50)2 - 4 × [Io] × [Eo]]1/2}; [Io] ) inhibitor

concentration; [Eo] ) enzyme concentration ) 1 nM, using the Kaleidagraph software package (Synergy Software, USA).

RESULTS We have constructed a library of 8000 DNA-tagged benzamidine inhibitors based on the stepwise addition of two building blocks onto a core structure consisting of H-Glu-(4-(aminomethyl)benzamidine)-(4-(aminomethyl)benzamidine) (Scheme 1). This building block was initially coupled to 40 Fmocprotected amino acids, and for each derivative, the side-chain carboxylic group of glutamate was deprotected and subsequently coupled to individual oligonucleotides carrying a primary amino group at the 5′ extremity. This reaction step allowed the DNAencoding of the first building block (13, 15, 16). After Fmoc deprotection and HPLC purification (Supporting Information Table 1), the 40 oligonucleotide conjugates were pooled and split into 200 reaction vessels, where the primary amino group was covalently modified with carboxylic acids, isocyanates, sulfonyl chlorides, or cyclic anhydrides (Scheme 1). At the end of this procedure, which was already known from model reactions on oligonucleotide-tagged amines to proceed with good yields and selectivity (Supporting Information Figure 1), the second building block was encoded by hybridization of partially complementary oligonucleotides carrying an eight-base code (Scheme 1). Double-stranded DNA fragments were obtained using Klenow DNA polymerase, in full analogy to what was previously reported by our group (13, 15, 16). Fully encoded

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Figure 2. Characterization of hits from trypsin selection. (a,b) Correlation of the high-throughput sequencing enrichment factor and trypsin inhibition constant. Individual compounds identified in the trypsin selection at 0.02 mg/mL protein coating density were synthesized as glutamine derivatives and incubated at different concentrations with bovine pancreatic trypsin. The reaction was started by the addition of the fluorogenic substrate Z-GGR-AMC and the change of fluorescence signal was recorded. Inhibition constants were determined by fitting. The 18 compounds are ordered on the x-axis from left to right according to their enrichment factor. The enrichment factor was derived by dividing the high-throughput sequencing counts of a specific code after trypsin selection by the counts obtained after selection on empty resin (see Supporting Information Figure 2). (c) Table summarizing the inhibition constants of the 21 synthesized compounds for 4 different serine proteases (trypsin, thrombin, uPA, and FXa). Three of the best trypsin inhibitors were also synthesized as alanine derivatives in order to investigate the contribution of the glutamine linker to the inhibition. The structures of the two building blocks forming the BAM8000 library compounds can be found in Supporting Information Tables 1 and 2, respectively.

library members were purified on chromatographic cartridges, pooled, and aliquoted for library use. The library of 8000 benzamidine derivatives was panned on sepharose resins covalently modified with a series of concentrations of trypsin (0.5, 0.1, 0.02, 0.004, and 0 mg trypsin per milliliter of CNBr-activated sepharose resin). After extensive washing, the DNA-tags of the library compounds that remained attached to the resins were used as a template for PCR amplification and the resulting amplicon mixtures were characterized by high-throughput DNA sequencing, using 454 technology (22) (15242, 15966, 17558, 15488, and 17145 counts, respectively, could be evaluated), in order to assess the relative abundance of individual codes for the library compounds after selection under different conditions (13-16). Figure 1 shows that the selection performed on 0.02 mg/mL trypsin coated resin yielded the best discrimination among library compounds. Indeed, it is conceivable that at high coating density all library members may efficiently be captured with the aid of the benzamidine moiety, while at low coating density, overall capture efficiency may be insufficient even for the highest-affinity binders. Similar observations have been previously made in the field of phage display library biopanning (23-28), as well as in the screening of DNA-encoded chemical libraries (9).

For the first building block, moieties 21 and 22 (a diphenylmethyl and a phenylsulfonamide group, respectively) dominated the trypsin selection experiments, while various groups corresponding to the second building block were selected. Representative structures indicating some of the most frequently enriched compounds are displayed in Figure 1. Figure 2a presents the list of the 12 most enriched compounds (sorted according to their enrichment factor, calculated as a ratio of sequence counts after selection on 0.02 mg/mL resin and after selection on empty resin; see also Supporting Information Figure 2). In addition, 6 non-enriched compounds are also considered for the comparison. The 18 compounds were resynthesized in the absence of DNA as glutamine derivatives (the most similar BAM8000 library format) and were characterized in terms of their 50% inhibitory concentration (IC50) of the catalytic activity of trypsin, using a fluorogenic substrate (11). In general, preferentially enriched compounds exhibited better IC50 values compared to the nonenriched counterpart (Figure 2b). The correlation between enrichment and potency was not 1:1, as other factors (e.g., reaction yields, nonspecific interactions with the resin support) may contribute to a selection bias (23-25). However, 6 out of 12 of the preferential binders exhibited an IC50 value of 1000-fold for trypsin, compared to the other three serine proteases.

DISCUSSION Three main lessons could be learned from this affinitymaturation project. First, libraries containing thousands of members could be rapidly synthesized with a “split and pool” approach, which allowed the unambiguous identification and quantification of individual library members with the help of the tagging with distinctive DNA fragments serving as amplifiable identification bar codes. Second, affinity selections were best performed using a variety of experimental conditions (e.g., densities of trypsin coating on resin), in order to identify the best preferential binders and minimize resynthesis work after library selections. Third and most important, a benzamidine lead compound (which displays trypsin IC50 values in the 100 µM concentration range; Figure 2c) could be affinity-matured to single-digit nanomolar potency, with an excellent discrimination toward related serine proteases. We anticipate that similar affinity maturation strategies can now be routinely implemented with leads against a variety of targets of pharmaceutical interest.

ACKNOWLEDGMENT This work was supported by Philochem AG, the Kommission fu¨r Technologie und Innovation (KTI Grant Nr. 8868.1 PFDSLS) and the Gebert-Ru¨f Foundation (Grant Nr. GRS-076/06) is gratefully acknowledged. The authors declare conflict-of-interest. D.N. owns shares of Philochem AG and J.S. consults for this company. Supporting Information Available: Detailed experimental procedure, library synthesis, high-throughput sequencing identification, enzyme inhibition assays, Supplementary Figures and Supplementary Tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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