A DNA-Encoded Library of Chemical Compounds Based on Common Scaffolding Structures Reveals the Impact of Ligand Geometry on Protein Recognition

Abstract

A DNA-encoded chemical library (DECL) with 1.2 million compounds was synthesized by combinatorial reaction of seven central scaffolds with two sets of 343×492 building blocks. Library screening by affinity capture revealed that for some target proteins, the chemical nature of building blocks dominated the selection results, whereas for other proteins, the central scaffold also crucially contributed to ligand affinity. Molecules based on a 3,5-bis(aminomethyl)benzoic acid core structure were found to bind human serum albumin with a K_d value of 6 nm, while compounds with the same substituents on an equidistant but flexible l-lysine scaffold showed 140-fold lower affinity. A 18 nm tankyrase-1 binder featured l-lysine as linking moiety, while molecules based on d-Lysine or (2S,4S)-amino-l-proline showed no detectable binding to the target. This work suggests that central scaffolds which predispose the orientation of chemical building blocks toward the protein target may enhance the screening productivity of encoded libraries.

Keywords: DNA-encoded chemical libraries; bifunctional ligands; human serum albumin; tankyrase-1.

Figure 1

Representation of the library synthesis scheme. Seven building blocks (1-7), carrying protected amine functions and azides, were coupled to amino-tagged oligonucleotides, each containing a central sequence (“code”), which unambiguously identifies the corresponding building block. After deprotection and using a split and pool procedure, carboxylic acids were coupled to the amines and the corresponding 343 building blocks “B” encoded by a ligation procedure. In a last synthesis step the azide moiety of the central scaffolds was either reacted with terminal alkynes or converted to a primary amine, allowing the formation of an amide bond with carboxylic acids. The final encoding step for building blocks “C” was performed using partially complementary oligonucleotides and Klenow polymerization, as described[6].

Figure 2

Results of library selections against human serum albumin (HSA). (a) Selection fingerprint. The individual library members are unambiguously identified by their code A (ranging between 1 and 7), B (ranging between 1 and 343) and C (ranging between 1 and 492). The number of sequence counts for each compound is displayed as spheres of a different colour, with a cut-off threshold set at 3000 counts. In total, 59,596,519 sequence counts were read by high-throughput sequencing. (b) The Code A analysis shows how the different scaffolds (code A, ranging 1 and 7) display different enrichment factors for each building block A and B combination (Code B × Code, ranging between 1 and 1.7×10⁵). (c) Selection fingerprint obtained fixing the code B = 66 (corresponding to 3-(3,4,5-trimethoxyphenyl)propanoic acid) with variable codes A (ranging between 1 and 7) and C (ranging between 1 and 492). This analysis shows the A7/B66/C292 as the most enriched combination of building blocks against HSA. (d) Fluorescence polarization (FP) measurement of FITC-conjugates of the most enriched combination of building blocks (B66/C292), featuring the preferred scaffold A7 (red and blue curves) or scaffolds A4 (green) and A6 (orange). A FITC conjugate of A7/B66/C292 was also tested against bovine serum albumin (BSA) a d revealed a double-digit micromolar dissociation constant (K_d) against that protein (pink curve).

Figure 3

Binding properties of compound A7/B66/C292 analyzed by Surface Plasmon Resonance (SPR) against HSA immobilized on a BIAcore chip (CM5, 5770 RU). (a) SPR profile of compound R-(A7/B66/C292) at different concentration (5 μM, 1.25 μM, 312 nM, 78.1 nM, 19.5 nM). An overall fitting of the resulting curves (4.7μM, 2.3 μM, 1.2 μM and 582 nM) revealed a k_off = 7.4x10^-3 Ms^-1 and a k_on = 2.0x10⁵ s^-1M^-1, resulting in a K_d = 36.6 nM (b) SPR profile of compound R-(A6/B66/C292) at different concentrations (5 μM, 1.25 μM, 312 nM, 78.1 nM, 19.5 nM). (c) SPR profile of compound R-(A4/B66/C292) at different concentrations (5 μM, 1.25 μM, 312 nM, 78.1 nM, 19.5 nM).

Figure 4

Results of library selections against human tankyrase-1 (TNKS1). (a) Selection fingerprint, reavealing a preferential enrichment of compounds with A4 and B101. (b) Plot of sequence counts for library members, featuring B101 as preferred building block. The plot reveals a preferential enrichment of library members with linker A4 and building blocks C491, C453, C369, C183 and C182. The structures of the four most enriched compounds is shown in panel (c). The BIAcore profiles at various concentrations of ligand for the amide derivatives of A4/B101/C491, A3/B101/C491 and A5/B101/C491 are shown in panels (d), (e) and (f). Fitting of the sensograms for A4/B101/C491 in panel (d) yielded a k_off = (2.1±0.8) x 10^-3 s^-1 and a k_on = (1.7±0.2) x 10⁵ s^-1M^-1, corresponding to a K_d = 15±8 nM.

Figure 5

Results of library selections against carbonic anhydrase IX (CAIX). (a) Selection fingerprint. The individual library members are unambiguously identified by their code A (ranging between 1 and 7), B (ranging between 1 and 343) and C (ranging between 1 and 492). The number of sequence counts for each compound is displayed as spheres of different colors, with a cut-off threshold set at 3000 counts. In total, 59,596,519 sequences were read by high-throughput DNA sequencing. (b) Selection fingerprint obtained fixing the code C = 410 (corresponding to acetazolamide) with variable codes A (ranging between 1 and 7) and B (ranging between 1 and 343). This picture shows how the scaffold does not affect the affinity for CAIX.