A Specific and Covalent JNK-1 Ligand Selected from an Encoded Self-Assembling Chemical Library

Abstract

We describe the construction of a DNA-encoded chemical library comprising 148 135 members, generated through the self-assembly of two sub-libraries, containing 265 and 559 members, respectively. The library was designed to contain building blocks potentially capable of forming covalent interactions with target proteins. Selections performed with JNK1, a kinase containing a conserved cysteine residue close to the ATP binding site, revealed the preferential enrichment of a 2-phenoxynicotinic acid moiety (building block A82) and a 4-(3,4-difluorophenyl)-4-oxobut-2-enoic acid moiety (building block B272). When the two compounds were joined by a short PEG linker, the resulting bidentate binder (A82-L-B272) was able to covalently modify JNK1 in the presence of a large molar excess of glutathione (0.5 mm), used to simulate intracellular reducing conditions. By contrast, derivatives of the individual building blocks were not able to covalently modify JNK1 in the same experimental conditions. The A82-L-B272 ligand was selective over related kinases (BTK and GAK), which also contain targetable cysteine residues in the vicinity of the active site.

Keywords: DNA-encoded chemical libraries; covalent inhibitors; drug discovery; kinases; targetable cysteine.

Figure 1. Design and encoding of the dual-display DNA-encoded chemical library.

(A) Overview of library construction. The synthesis of sub-library A (red) was performed by coupling a chemical fragment to an oligonucleotide with a distinct coding sequence that unambiguously identifies each individual library member. The conjugates were individually purified and characterized by LC-MS. The sub-library A is composed of 265 members, of which 42 are capable of covalent bond formation. The synthesis of sub-library B (blue) has been described previously (1, Wichert) and was extended with additional 359 building blocks, of which 114 are capable of covalent bond formation. With the help of an adapter, the conjugates were encoded by ligation to code B oligonucleotides. Sub-library B was hybridized with sub-library A and Klenow polymerase-assisted fill-in reaction allowed the code transfer onto the sub-library A strand to form the final library of 148’135 members. (B) Schematic representation of a member of the dual-display library binding to a target protein. The library is formed by hybridization of two individually synthesized sub-libraries A and B, resulting in a combinatorial library A × B.

Figure 2. Selection results from the 148’135-member dual-display library.

High-throughput DNA sequencing (HTDS) plot of selections against JNK1. The x–y plane represents library member barcodes of sub-library A and sub-library B, and the z axis shows the sequence counts (cutoff level = 200). The most enriched fragment pair corresponds to A82 (in sub-library A) and B272 (in sub-library B).

Figure 3. Mass analysis assay for covalent JNK1 binder.

(A) Schematic overview of the MS analysis assay for the detection of covalently modified JNK1 protein in the presence or absence of glutathion. (B) The most enriched fragment pair A82 and B272 connected with a short polyethyleneglycol-linker A82-L-B272 and the compound B-272 modified with a simple propylamine used as a monovalent control compound B272-PA. (C) A82-L-B272 treated JNK1 kinase domain. Mass spectra were obtained after deconvolution and show addition of 539 Daltons after incubation with A82-L-B282. Assay conditions: 0.4 µM JNK1 in 50 mM HEPES, 300 mM NaCl, 10 mM MgCl₂, 1 µM TCEP; 0.5 mM glutathion; 3.0 µM A82-L-B282. (D) Hit validation of selected building block pair against JNK1, BTK and GAK. By MS analysis, monovalent A82 and B272-PA show no detectable (n.d.) JNK1 modification. Assay conditions: 0.4 µM JNK1/GAK in 50 mM HEPES, 300 mM NaCl, 10 mM MgCl₂, 1 µM TCEP; 0.4 µM BTK in 20 mM Tris, 240 mM NaCl, 30 µM DTT; +/− glutathion (GSH, 0.5 mM); 1.6 µM binder. Error bars indicate standard deviations of three independent measurements. (E) Titration of JNK1 with increasing concentration of binder A82-L-B272. Titration conditions: 0.4 µM JNK1 in 50 mM HEPES, 300 mM NaCl, 10 mM MgCl₂, 1 µM TCEP; glutathion (GSH, 0.5 mM); 0.1 - 4.0 µM binder A82-L-B272. Error bars indicate deviations of two independent measurements.