DNA-Encoded Chemical Libraries for the Discovery of MMP-3

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Bioconjugate Chem. 2008, 19, 778–785

DNA-Encoded Chemical Libraries for the Discovery of MMP-3 Inhibitors Jörg Scheuermann, Christoph E. Dumelin,† Samu Melkko,† Yixin Zhang, Luca Mannocci, Madalina Jaggi, Jens Sobek,‡ and Dario Neri* Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland. Received November 27, 2007; Revised Manuscript Received December 3, 2007

Encoded self-assembling chemical (ESAC) libraries are characterized by the covalent display of chemical moieties at the extremity of self-assembling oligonucleotides carrying a unique DNA sequence for the identification of the corresponding chemical moiety. We have used ESAC library technology in a two-step selection procedure for the identification of novel inhibitors of stromelysin-1 (MMP-3), a matrix metalloproteinase involved in both physiological and pathological tissue remodeling processes, yielding novel inhibitors with micromolar potency.

INTRODUCTION Chemical compounds carrying unique DNA fragments as amplifiable identification “bar codes” may serve as building blocks for the construction and screening of chemical libraries of unprecedented size, with considerable implications for drug discovery (1–7). DNA-encoded chemical libraries can be classified as “single pharmacophore” chemical libraries (in which a single chemical structure is covalently attached to a DNA fragment) and as “dual pharmacophore” chemical libraries (in which each complementary DNA strand carries a chemical moiety) (Figure 1) (6). In the latter case, the two pharmacophores can simultaneously contact the target protein of interest, thus conferring a high binding affinity by virtue of the chelate effect (4, 8, 9). Dual pharmacophore DNA-encoded chemical libraries (ESAC1 libraries) may be used for the de novo identification of pairs of synergistic binding molecules to protein targets of interest or for the affinity maturation of known lead compounds, which can eventually be converted into small organic molecules devoid of the DNA portion. The process relies on the preferential enrichment of library compounds binding to the target protein of interest immobilized on a solid support, followed by PCR amplification of the recovered oligonucleotide-compound conjugates and by bar code identification on DNA microarrays (Figure 2) (4, 10). Matrix metalloproteinases (MMPs) are zinc-dependent proteases which are involved in tissue remodeling for a variety of physiological and pathological processes (11–14). More than 50 MMP inhibitors have entered clinical trials, 46 of which have been discontinued while 7 are still in the evaluation process (15). It is generally accepted that future endeavors for the * Corresponding author. Dario Neri, Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich, Switzerland. neri@ pharma.ethz.ch, Fax: +41 633 13 58, Tel: +41 633 74 01. † Philochem AG, c/o ETH Zürich, Zürich, Switzerland. ‡ Functional Genomics Center Zurich, ETH Zurich/ University of Zurich, Zurich, Switzerland. 1 Abbreviations: CAII, carbonic anhydrase II; CPA, carboxypeptidase A; DMSO, dimethyl sulfoxide; EDC, 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide; ESAC library, encoded self-assembling chemical library; hu-MMP3, human MMP-3; mu-MMP3, murine MMP-3; PBS, phosphate buffered saline; TBS, Tris-buffered saline, TEA, triethylamine; SSC, sodium chloride-sodium citrate; uPA, urokinase-type plasminogen activator.

Figure 1. Schematic representation of different formats of encoded self-assembling chemical (ESAC) libraries which can be used for selections against a target antigen. Small molecules are coupled to oligonucleotides, which contain a hybridization domain and (if appropriate) a code revealing the identity of the covalently coupled molecule. After DNA-duplex formation, the oligonucleotide-compound conjugate sublibraries can be used in single pharmacophore selections (A), affinity maturation selections of known binders (B), or de novo selections of binding molecules from a combinatorial library generated by assembling two sublibraries (C).

Figure 2. A typical ESAC library selection and readout procedure. The DNA-duplex library (a) is panned against the target protein of interest (b). After washing and removal of unbound molecules (c), the codes of the remaining binders are PCR amplified and compared with the unselected library on oligonucleotide microarrays (d,e). Resulting binding pairs are then validated and positive binding partners are conjugated to suitable scaffolds yielding small druglike molecules (f).

pharmaceutical targeting of MMPs should aim at the development of more selective inhibitors, and in the identification of those MMPs which play a crucial role in pathogenesis (13, 16, 17). In this article, we present the use of ESAC libraries and technologies for both the identification of a novel MMP-3 inhibitory scaffold and its ESAC technology-assisted affinity maturation, yielding micromolar MMP-3 inhibitors.

EXPERIMENTAL PROCEDURES Cloning and Expression of Human MMP-3 Catalytic Domain. The gene of human MMP-3 catalytic domain (residues 100–272) (18) was subcloned into a pQE12 vector (Qiagen,

10.1021/bc7004347 CCC: $40.75  2008 American Chemical Society Published on Web 02/07/2008

DNA-Encoded Chemical Libraries Table 1. Compounds Derived from MMP3 Selections

Scheme 1. Synthesis of Compounds Linking Pharmacophores I and IIa

a (I) Linkage of I precursor carboxylic acid by carbodiimide-mediated activation with a diamino linker. (II) Coupling of I-amine with II precursor isothiocyanate to a dual pharmacophore compound.

Hombrechtikon, Switzerland) using restriction sites EcoRI and BglII and the primers 5′-ACT GAA TTC ATT AAA GAG GAG AAA TTA ACT ATG TTC AGA ACC TTT CCT GGC ATC3′ and 5′-ACT AGA TCT CTA TCA GTG ATG GTG ATG GTG ATG GCC ACC GGG GGT CTC AGG GGA GTC AG-3′. The protein containing a C-terminal His6-tag was purified with a NiNTA column (Qiagen) and dialyzed against 50 mM Tris-Cl, 200 mM NaCl, pH 7.3, and stored frozen at -20 °C. Cloning and Expression of Murine MMP-3 Catalytic Domain. The murine MMP-3 catalytic domain (residues 100–273) was isolated from a murine cDNA library (eye) and cloned into a pQE12 vector (Qiagen, Hombrechtikon, Switzerland) using restriction sites EcoRI and BglII and the primers 5′- CCG GAA TTC ATT AAA GAG GAG AAA TTA ACT ATG TTC AGT ACC TTC CCA GG-3′ and 5′-ACT AGA TCT CTA TCA GTG ATG GTG ATG GTG ATG GCC ACC GAG GAC ATC AGG GGA TGC TGT-3′. The protein containing a C-terminal His6-tag was purified with a Ni-NTA column (Qiagen) and dialyzed against 50 mM Tris-Cl, 200 mM NaCl, pH 7.3, and stored frozen at -20 °C. Synthesis of Oligonucleotide Conjugates and Linked Pharmacophores. All chemicals were purchased from SigmaAldrich-Fluka (Buchs, Switzerland), unless otherwise denoted. The synthetic principles of the DNA-encoded chemical library of 550 compounds have been described before (10). A list of the 550 oligonucleotide-compound conjugates can be found in the Supporting Information (Table 1). In short, 48mer oligonucleotides (IBA GmbH, Göttingen, Germany), carrying a free amino group at the 5′ end (as ω-aminohexyl phosphate diester) were used of the general structure 5′-GGA GCT TCT GAA TTC TGT GTG CTG XXX XXX CGA GTC CCA TGG CGC AGC-3′, where the XXX XXX coding sequence unambiguously codes for the individual chemical compound displayed. The chemical compounds

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comprising carboxylic acid, carboxylic anhydride, N-hydroxysuccinimide ester, or isothiocyanate groups were coupled to the primary amino group of the oligonucleotides (4, 10). Selection for Binding. The library containing each individual member at a concentration of 1.78 nM was dimerized at a concentration of 100 nM by heteroduplex formation with an unmodified pairing oligonucleotide (for de novo selections) or with a 3′-amino-modified oligonucleotide, which was conjugated to 3-(4-(cyclohexylmethoxy)phenyl) propanoic acid by an amide bond (for affinity maturation selections). Human MMP-3 and control resins were prepared as follows: 317 mg of dry CNBrmodified sepharose (GE Healthcare) were thoroughly washed with cold 1 mM HCl and incubated overnight at 4 °C either with 4 mL of 50 µM MMP-3 in coupling buffer (100 mM NaHCO3, 100 mM NaCl, pH 8.1) or with 1 M Tris-Cl, pH 7.2 (control resin). The MMP-3 resin was washed 5 times with 550 µL of coupling buffer and excess reactive groups on the resin were quenched with 550 µL of 1 M Tris-Cl, pH 7.2, at 4 °C overnight. The resins were then washed at alternating pH with 100 mM sodium acetate, 500 mM NaCl, pH 4.7, and 100 mM Tris-Cl, 500 mM NaCl, pH 8.1 (4 times each) using SpinX columns (Corning Life Sciences, Acton, USA) and resuspended in 2 mL of selection buffer (50 mM Tris-Cl, 200 mM NaCl, 10 mM CaCl2, 1 mM MgCl2, 10 µM ZnCl2, pH 6.7) containing 0.05 mg/mL herring sperm DNA. 10 µL of the DNA duplex library (85 nM total concentration) was then incubated with 200 µL of resin and 200 µL of selection buffer. After incubation for 1 h at 25 °C, the suspension was transferred to a SpinX column (Corning), the supernatant removed, and the resin washed 3 times with 400 µL of selection buffer. The resin was finally resuspended in 400 µL of selection buffer and stored at -20 °C. Decoding. The codes of the oligonucleotide-compound conjugates were amplified after selection by PCR (50 µL, 25 cycles of 1 min at 94 °C, 1 min at 55 °C, 40 s at 72 °C), using 10 µL of the resuspended resin as template and primers AB_fo_short (GGA GCT TCT GAA TTC TGT GTG CTG) and Elib2_ba (GCT GCG CCA TGG GAC TCG). The product was purified with a PCR purification kit (Qiagen) and subsequently linearly amplified by PCR (as above) using 5′-Cy3labeled Elib2_ba as primer. The product was precipitated by adding 10% v/v of 3 M sodium acetate, pH 4.7, and 250% v/v of ethanol and redissolved in 120 µL of hybridization buffer (4 × SSC (60 mM sodium citrate, 0.6 M NaCl, pH 7), 50 mM HEPES, 0.2% SDS, pH 7) and hybridized on microarray glass slides (displaying 19-mers with the structure 5′-T GTG CTG XXX XXX CGA GTC-3′) in a Tecan HS 400 instrument for 4 h at 44 °C, followed by successive washing steps with 2 × SSC/0.2% (w/v) SDS, 0.2 × SSC/0.2% (w/v) SDS, and 0.2 × SSC. After hybridization, the microarrays were analyzed by scanning with a Genepix professional 4200A scanner (Molecular Devices, Sunnyvale, CA; λex ) 532 nm, 100% laser power, photomultiplier 400). Spot intensities were quantified and evaluated using Genespotter software (v 2.4.3, MicroDiscovery, Berlin, Germany). After background subtraction, the mean value was used as spot signal intensity (average of all spots of the same oligonucleotide). Dividing the obtained signal intensities for the MMP-3 selection by the intensities of the control selection (no antigen resin) resulted in a fingerprint of the enrichment profile. Affinity Chromatography of Enriched ESAC Conjugates. Oligonucleotide 5′-CAG CAC ACA GAA TTC AGA AGC TCC-3′ and the oligonucleotide conjugate 5′-CAG CAC ACA GAA TTC AGA AGC TCC-I-3′ were radioactively labeled at the 5′ terminus using γ33P-ATP (GE Healthcare) and T4 polynucleotide kinase (USB, Cleveland, OH) and individually hybridized to enriched ESAC library compounds or controls

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Table 2. Inhibition Constants of Dual Pharmacophore Conjugates Derived from MMP3 Selection

at 200 nM final concentration. 15 µL of the hybridization was incubated with 35 µL of selection buffer and 50 µL MMP-3 resin (see above). After 45 min incubation at 25 °C, the resin was washed several times with 400 µL of selection buffer on SpinX columns (Corning). Aliquots of the remaining resin, input, flowthrough, and all washing fractions were subjected to 33 P radioactivity counting on a Beckman LS6500 scintillation counter (Beckman Coulter, Fullerton, CA). Linked Pharmacophores. The synthesis of bidentate conjugates of I and II was performed according to Scheme 1: 3-(4(cyclohexylmethoxy)phenyl) propanoic acid (20 µmol) was activated for 30 min at 25 °C with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (50 µmol) and N-hydroxy succinimide (NHS) (100 µmol) in DMSO and subsequently reacted with diaminolinker (200 µmol)/TEA (144 µmol) overnight at 30 °C. The resulting linker conjugate was purified by HPLC, dried, redissolved in 200 µL of DMSO, and reacted after addition of TEA (72 µmol) with 30 µmol of 4-acetamido-4′-

isothiocyanostilbene-2,2′-disulfonic acid. After quenching with a molar excess of ethanolamine, the reaction was purified by HPLC, dried, and analyzed by ESI-MS (Quattro Micro, Waters, Milford, CT). 1H NMR spectra were measured on a Bruker 400 avance instrument (Bruker, Ettlingen, Germany) and concentrations determined using nitromethane as internal standard (compound naming R-Linker-R′: cf. Tables 1 and 2). I-A-II.(E)-5-Acetamido-2-(4-(3-(2-(3-(4-(cyclohexylmethoxy) phenyl)propanamido)ethyl)thioureido)-2-sulfostyryl)benzenesulfonic acid. ESI-MS (m/z): 759.2 ([M + H+], 100%), calcd: 758.21 Da. 1H NMR (DMSO-d6): 7.95 (d, 2.5 Hz, 2H), 7.71 (d, 2.5 Hz, 2H), 7.69 (d, 2.5 Hz, 1H), 7.57–7.47 (m, 3H), 7.09 (d, 8.5 Hz, 2H), 6.80 (d, 8.5 Hz, 2H), 3.71 (d, 6.3 Hz, 2H), 3.24 (m, 2H), 2.74 (t, 7.8 Hz, 2H), 2.40–2.32 (m, 4H), 2.04 (s, 3H), 1.82–1.60 (m, 6H), 1.35–0.96 (m, 5H). I-B-II. (E)-5-(3-(4-(3-(4-(Cyclohexylmethoxy)phenyl)propanamido)butyl)thioureido)-2-(4-ethanamido-2-sulfostyryl)benzenesulfonic acid. ESI-MS (m/z): 787.2 ([M + H+], 100%),

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Figure 3. De novo selection of MMP-3 binders using a DNA duplex library consisting of a 3′-unmodified oligonucleotide hybridized to an ESAC library of 550 compounds. (A) Microarray readout after PCR amplification of a selection against MMP-3-displaying resin (upper panel) and control resin (no antigen, lower panel). On the microarrays, ESAC library decoding oligonucleotides are spotted in quadruplicate. The spots encoding the enriched ESAC oligonucleotide-compound conjugate 327 are shown as insets for both MMP-3 and control selection. (B) Quantitative microarray evaluation: Enrichment of ESAC library oligonucleotide-compound conjugates given as ratio of MMP-3 selection versus control selection. The enriched ESAC conjugate 327 is indicated. (C) Affinity chromatography of enriched ESAC conjugate 327 and of nonenriched ESAC conjugate 2 (- ctrl) hybridized each to a radiolabeled unmodified oligonucleotide. The retaining radioactivity was monitored for increasing washing steps.

calcd: 786.24 Da. 1H NMR (partial, DMSO-d6): 3.72 (d, 6.3 Hz, 2H, R-OCH2-cyclohexyl of I), 2.04 (s, 3H, CH3-CONHAr of II). I-C-II. (E)-5-(3-(6-(3-(4-(Cyclohexylmethoxy)phenyl)propanamido)hexyl)thioureido)-2-(4-ethanamido-2-sulfostyryl)benzenesulfonic acid. ESI-MS (m/z): 815.2 ([M + H+], 100%), calcd: 814.27 Da. 1H NMR (partial, DMSO-d6): 3.72 (d, 6.3 Hz, 2H, R-OCH2-cyclohexyl of I), 2.04 (s, 3H, CH3-CONHAr of II). I-D-II. (E)-5-(3-(17-(4-(Cyclohexylmethoxy)phenyl)-15-oxo4,7,10-trioxa-14-azaheptadecyl)thioureido)-2-(4-ethanamido-2sulfostyryl)benzenesulfonic acid. ESI-MS (m/z): 919.3 ([M + H+], 100%), calcd: 918.32 Da. 1H NMR (partial, DMSO-d6): 3.72 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl of I), 2.04 (s, 3H, CH3-CONHAr of II). I-D-III. N-(17-(4-(Cyclohexylmethoxy)phenyl)-15-oxo-4,7,10trioxa-14-azaheptadecyl)-2,6-difluorobenzamide. ESI-MS (m/z): 605.4 ([M + H+], 100%), calcd: 604.33 Da. 1H NMR (partial, DMSO-d6): 7.50 (t, 7.5 Hz, 1H, p-H of III), 3.71 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl of I). I-D-NH2. N-(3-(2-(2-(3-Aminopropoxy)ethoxy)ethoxy)propyl)-3-(4-(cyclohexylmethoxy)phenyl)propanamide. ESI-MS (m/z): 465.4 ([M + H+], 100%), calcd: 464.33 Da. 1H NMR (partial, DMSO-d6): 3.72 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl). II-D-NH2.(E)-5-(3-(3-(2-(2-(3-Aminopropoxy)ethoxy)ethoxy) propyl)thioureido)-2-(4-ethanamido-2-sulfostyryl)benzenesulfon-

ic acid. ESI-MS (m/z): 675.3 ([M + H+], 100%), calcd: 674.18 Da. 1H NMR (partial, DMSO-d6): 2.04 (s, 3H, CH3-CONHAr). I-E-II. (E)-5-(3-(3-(4-(3-(3-(4-(Cyclohexylmethoxy)phenyl)propanamido)propyl)piperazin-1-yl)propyl)thioureido)-2-(4ethanamido-2-sulfostyryl)benzenesulfonic acid. ESI-MS (m/z): 899.3 ([M + H+], 100%), calcd: 898.34 Da. 1H NMR (partial, DMSO-d6): 3.72 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl of I), 2.04 (s, 3H, CH3-CONHAr of II). I-F-II. (E)-5-(3-((3-((3-(4-(Cyclohexylmethoxy)phenyl)propanamido)methyl)cyclohexyl)methyl)thioureido)-2-(4-ethanamido2-sulfostyryl)benzenesulfonic acid. ESI-MS (m/z): 841.2 ([M + H+], 100%), calcd: 840.29 Da. 1H NMR (partial, DMSOd6): 3.71 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl of I), 2.04 (s, 3H, CH3-CONHAr of II). I-G-II. (E)-5-(3-((4-((3-(4-(Cyclohexylmethoxy)phenyl)propanamido)methyl)cyclohexyl)methyl)thioureido)-2-(4-ethanamido2-sulfostyryl)benzenesulfonic acid. ESI-MS (m/z): 841.2 ([M + H+], 100%), calcd: 840.29 Da. 1H NMR (partial, DMSOd6): 3.71 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl of I), 2.04 (s, 3H, CH3-CONHAr of II). I-H-II. (E)-5-(3-(3-((3-(4-(Cyclohexylmethoxy)phenyl)propanamido)methyl)benzyl)thioureido)-2-(4-ethanamido-2-sulfostyryl)benzenesulfonic acid. ESI-MS (m/z): 835.3 ([M + H+], 100%), calcd: 834.24 Da. 1H NMR (partial, DMSO-d6): 3.72 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl of I), 2.04 (s, 3H, CH3CONHAr of II).

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Figure 4. Affinity maturation selection of ESAC conjugate 327 (I) using a DNA duplex library consisting of an oligonucleotide displaying I on its 3′ terminus hybridized to an ESAC library of 550 compounds. (A) Microarray readout after PCR amplification of affinity maturation selection against MMP-3-displaying resin (upper panel) and control resin (no antigen, lower panel). On the microarrays, ESAC library decoding oligonucleotides are spotted in quadruplicate. The spots encoding the enriched ESAC library conjugate 581 are shown as insets for both MMP-3 and control selection. (B) Quantitative microarray evaluation: Enrichment of ESAC library conjugates given as ratio of MMP-3 selection versus control selection. The enriched ESAC conjugate 581 is indicated. (C) Affinity chromatography of ESAC conjugates 327 and 581 hybridized to an unmodified oligonucleotide (OH/581 and 327/OH), of the combination of both pharmacophores (327/581) and of a unmodified oligonucleotide heteroduplex control (OH/OH). The 5′-radiolabeled oligonucleotide is marked with an asterisk. The retaining radioactivity was monitored for increasing washing steps.

I-J-II. (E)-5-(3-(4-((3-(4-(Cyclohexylmethoxy)phenyl)propanamido)methyl)benzyl)thioureido)-2-(4-ethanamido-2-sulfostyryl)benzenesulfonic acid. ESI-MS (m/z): 835.1 ([M + H+], 100%), calcd: 834.24 Da. 1H NMR (partial, DMSO-d6): 3.72 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl of I), 2.04 (s, 3H, CH3CONHAr of II). I-J-III. N-(4-((3-(4-(Cyclohexylmethoxy)phenyl)propanamido)methyl)benzyl)-2,6-difluorobenzamide. ESI-MS (m/z): 521.3 ([M + H+], 100%), calcd: 520.25 Da. 1H NMR (partial, DMSOd6): 7.53 (t, 7.5 Hz, 1H, p-H of III), 3.73 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl of I). I-J-NH2. N-(4-(Aminomethyl)benzyl)-3-(4-(cyclohexylmethoxy)phenyl)propanamide. ESI-MS (m/z): 381.3 ([M + H+], 100%), calcd: 380.25 Da, 1H NMR (partial, DMSO-d6): 3.73 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl). I-K-COOH. 7-(3-(4-(Cyclohexylmethoxy)phenyl)propanamido)heptanoic acid. ESI-MS (m/z): 390.3 ([M + H+], 100%), calcd: 389.26 Da. 1H NMR (partial, DMSO-d6): 3.71 (d, 6.4 Hz, 2H, R-OCH2-cyclohexyl). MMP-3 Inhibition Assay. 55 µL of MMP-3 in 7.3 mM Tris-Cl, 36.4 mM NaCl, 63.6 mM 2-(N-morpholino)ethane sufonate, 1.27 mM CaCl2, 25.5 µM ZnCl2, 0.055% Brij-35, pH 6.1 (final MMP-3 concentration: 417 nM), were incubated in a 384-well microtiter plate (Nunc, Roskilde, Denmark) with a

dilution series of inhibitor in DMSO (5 µL) for 20 min at 25 °C. The reaction was started by addition of 10 µL of the fluorogenic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (OmniMMP, BIOMOL, Exeter, UK, dissolved at 50 µM in 5% DMSO) to 60 µL of MMP-3/inhibitor solution. The change of fluorescence signal (λex 328 nm; λem 393 nm) was recorded over 5 min using a SpectraMax Gemini microplate reader (Molecular Devices). The rate of fluorescence signal increase over time (initial reaction velocity V) was plotted against the corresponding inhibitor concentration, and the IC50 value for the inhibitor was obtained by fitting to the equation V ) R/2 × {-(I - Eo + IC50) + sqrt[(I - Eo + IC50)2 + 4 × IC50 × Eo]} (V ) initial velocity, Io ) initial inhibitor concn, Eo ) initial enzyme concn) using SigmaPlot software (SPSS Inc., Chicago, IL). uPA Inhibition Assay. 58 µL of 32 nM uPA in TBS were incubated in a 384-well microtiter plate (Nunc, Roskilde, Denmark) with a dilution series of inhibitor in DMSO (5 µL) for 20 min at 25 °C. The reaction was started by addition of 7 µL of the fluorogenic substrate ZGGR-AMC (1 mM in DMSO) (Bachem, Switzerland). The change of fluorescence signal (λex 383 nm; λem 455 nm) was recorded over 4 min using a SpectraMax Gemini microplate reader (Molecular Devices). The rate of fluorescence signal increase over time (initial reaction velocity V) was plotted against the corresponding inhibitor

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Figure 5. Verification of dual pharmacophore binding: Affinity maturation selection of ESAC conjugate 581 (II) using a DNA duplex library consisting of an oligonucleotide displaying II on its 3′ terminus hybridized to an ESAC library of 550 compounds. (A) Microarray readout after PCR amplification of an affinity maturation selection against MMP-3-displaying resin (left panel) and control resin (no antigen, right panel). On the microarrays, ESAC library decoding oligonucleotides are spotted in triplicate. The spots coding for the ESAC library conjugate 327 (I) are shown as insets for both MMP-3 and control selection. (B) Quantitative microarray evaluation: Enrichment of ESAC library conjugates given as ratio of MMP-3 selection versus control selection. The enriched ESAC library conjugate 327 is indicated.

concentration, and when possible, the IC50 value for the inhibitor was obtained by fitting to the above-mentioned equation using SigmaPlot software. CPA Inhibition Assay. 85 µL of 59 nM carboxypeptidase A (cat. no. C9268, Sigma, Buchs, Switzerland) in assay buffer (50 mM Tris-Cl, 1 M NaCl, 1 mM ZnCl2, pH ) 7.5) were incubated in a 96-well microtiter plate (Nunclon, Nunc, Roskilde, Denmark) with a dilution series of inhibitor in DMSO (5 µL) for 20 min at 25 °C. The reaction was started by addition of 10 µL of the substrate FA-Phe-Phe-OH (10 mM in DMSO) (cat. no. M1760, Bachem, Switzerland). The change in transmission at 340 nm was recorded over 10 min using a VersaMax microplate reader (Molecular Devices). The signal increase over time (initial reaction velocity V) was plotted against the corresponding inhibitor concentration, and when possible, the IC50 value for the inhibitor was obtained by fitting to the abovementioned equation using SigmaPlot software. CAII Inhibition Assay. 85 µL of 1.2 µg/mL carbonic anhydrase II (cat. no. C3934, Sigma, Buchs, Switzerland) in assay buffer (50 mM Tris-sulfate, 1 mM ZnCl2, pH ) 8.5) were incubated in a 96-well microtiter plate (Nunclon, Nunc, Roskilde, Denmark) with a dilution series of inhibitor in DMSO (5 µL) for 20 min at 25 °C. The reaction was started by addition of 10 µL of the substrate p-nitrophenyl acetate (10 mM in 1% acetonitril) (cat. no. M1760, Bachem, Switzerland). The change in absorbance at 405 nm was recorded over 10 min using a VersaMax microplate reader (Molecular Devices). The net signal increase over time (initial reaction velocity V) was plotted against the corresponding inhibitor concentration, and when possible, the IC50 value for the inhibitor was obtained by fitting to the above-mentioned equation using SigmaPlot software.

RESULTS In an attempt to find novel MMP-3 inhibitors, a library of 550 DNA-encoded chemical compounds was selected for binding to human MMP-3, encouraged by the recent observation that a precursor library of 477 oligonucleotide-compound conjugates could be successfully used in single pharmacophore selections (Figure 1A), yielding specific binders to streptavidin (10) and human serum albumin (30). Each DNA-encoded compound in the library was prepared by conjugating the chemical compound to the 5′ end of an amino-modified 48mer oligonucleotide carrying a 6-base code for identification

and regions for primer annealing and PCR amplification (10). Selection for MMP-3 binding was performed on human MMP-3 covalently coupled to CNBr-activated sepharose resin and on Tris-quenched CNBr-activated sepharose resin (control selection), similar to previously published procedures (4, 9). After thorough washing of the resin, the enriched DNA conjugates were PCR-amplified with a Cy3-labeled primer and hybridized to decoding microarrays (Figure 3A), carrying code-complementary oligonucleotides, spotted in quadruplicate. The ratio of the average fluorescence signal intensity corresponding to each library member from MMP-3 selection and control selection on control resin was determined, giving rise to a fingerprint of enriched binding molecules (Figure 3B). One compound (ESAC library conjugate 327, structure I of Table 1) was strongly enriched (7-fold) compared to background. This 327-oligonucleotide conjugate was then subjected to an affinity chromatography assay, displaying a substantial retention on MMP-3 resin compared to the control (nonenriched ESAC conjugate 2) (Figure 3C). In this assay, a 33P-labeled oligonucleotide was hybridized with the individual ESAC conjugates, incubated with MMP-3 resin and subjected to consecutive washing steps. The radioactivity retained on the resin after the washing steps provided a direct estimate of the MMP-3 binding ability of the individual DNA conjugates, a process which is known to be dependent on the protein density on resin (8–10). The newly discovered MMP-3 binder I was conjugated to the amino-modified 3′-extremity of a 24-mer oligonucleotide capable of pairing with the 550 member ESAC sublibrary and used as lead for affinity maturation selections (Figure 1B). After affinity capture on immobilized MMP-3, PCR amplification and microarray-based decoding (Figure 4A), enrichment of one synergistic binding moiety was observed (structure II of Table 1, corresponding to compound 581 in the ESAC library; Figure 4B). Preferential MMP-3 binding of the bidentate heteroduplex, displaying both compounds I and II, was confirmed in a radioactivity-based affinity chromatography assay performed as described above (Figure 4C). The bidentate binder was superior compared to DNA conjugates displaying the individual pharmacophores or no pharmacophore at all (Figure 4C). In order to confirm the results of the ESAC selection, affinity maturation selections were performed, using the DNA conjugate 581 (II) as a lead, paired to the 550-member ESAC library. As expected, the corresponding pharmacophore I was preferentially

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enriched by selections on MMP-3 resin, confirming the synergistic affinity gain offered by the combination of these two pharmacophores (Figure 5). The newly discovered pharmacophores I and II were used for the synthesis of low-molecular weight bidentate MMP-3 inhibitors with a series of diamino linkers (A-J, Table 2), according to Scheme 1. The corresponding inhibition constants to MMP-3 were measured, revealing that only the combination of the two pharmacophores yielded compounds of micromolar potency (Table 2). The highest binding affinity was observed for compound I-J-II, with an IC50 value of 9.9 µM. The compounds in the series were also characterized in terms of their IC50 values for the closely related murine MMP-3, for the Zn-dependent metalloproteinase carboxypeptidase A, for the human serine protease uPA, and for bovine carbonic anhydrase II. As expected, inhibitors of human MMP-3 were also able to inhibit the murine enzyme, but displayed only minor inhibitory activity toward both carboxypeptidase A and uPA, and no detectable activity toward carbonic anhydrase II (Table 2).

DISCUSSION The experiments presented in this article confirm that a library of 550 compounds, individually coupled to distinct oligonucleotides carrying an identification code, could be used for the identification of novel MMP-3 binding compounds, suitable for subsequent medicinal chemistry optimization. While monodentate MMP-3 binders did not exhibit any substantial inhibitory activity, bidentate inhibitors could be isolated with inhibitory constants in the low micromolar range (19–23). The linker connecting the two pharmacophores appeared to play only a moderate contribution to binding affinity, with IC50 values for bidentate binders ranging within a factor of 10. The bidentate binders were found to display a good selectivity toward the metalloprotease CPA, the serine protease (uPA) and toward carbonic anhydrase II (Table 2). The production of good-quality catalytic domains of other MMPs, required for the reliable profiling of inhibitory activities, however, remains a major challenge in the field, due to autoproteolytic processes (24). Most of the MMP-3 inhibitors reported so far in the literature featured a zinc-chelating group (e.g., hydroxamate, carboxylate, barbiturate, thiadiazole, thiadiazine, or sulfodiimine 14, 17, 25). While monodentate zinc-binding groups usually display IC50 values in the millimolar range, these moieties can be fused to a peptide mimetic group, thus enhancing both binding affinity and selectivity (26, 27). Barbiturate, carboxylates, and thiadiazole derivatives normally display micromolar inhibitory potency to MMP-3 (28), but occasionally higher potencies toward gelatinases (17). By contrast, hydroxamate derivatives with IC50 values for MMP-3 are ranging between 0.27 nM and > 1 µM (17). The present study confirms the potential of ESAC technology for the rapid identification of pairs of pharmacophores which, upon conjugation, display synergistic binding to the target protein of interest. Future directions of this line of research will require the construction of libraries of larger dimensions (e.g., >10 000 compounds) and the implementation of high-throughput decoding methodologies (e.g., by high-throughput sequencing 6, 29).

ACKNOWLEDGMENT We thank Dr. Christian Heinis and Stefanie Pfaffen for the construction of the human and murine MMP-3 expression vector, Catharine Aquino Fournier and Dr. Ralph Schlapbach from the Functional Genomics Center Zurich for help with microarray technology. Funding from ETH, SBF (EU Projects STROMA, FluorMMPI), the FP6 (EU Project Immuno-PDT), the Kommission für Technologie and Innovation, and the Swiss

Scheuermann et al.

National Science Foundation (Project 3100A0-105919) is gratefully acknowledged. Supporting Information Available: Supplementary table. This material is available free of charge via the Internet at http:// pubs.acs.org.

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