Affinity Selections of DNA-Encoded Chemical Libraries on Carbonic Anhydrase IX-Expressing Tumor Cells Reveal a Dependence on Ligand Valence.

DNA-encoded chemical libraries are typically screened against purified protein targets. Recently, cell-based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)-expressing tumor cells as a model system to optimize selection procedures with code-specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15-fold enrichment factors for high-affinity monovalent CAIX binders (acetazolamide; KD =8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100-fold). The optimized procedures were used for selections with a DNA-encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor-associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise.

On-DNA hit validation methodologies for ligands identified from DNA-encoded chemical libraries.

DNA-encoded chemical libraries (DECLs) are large compound collections attached to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of pharmaceutical interest. In DECL selections, ligands are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified using this procedure need to be validated by resynthesis and by performing affinity measurements. Here we report novel on-DNA hit validation strategies, which enable the facile confirmation of ligand-protein interaction as well as the determination of equilibrium and kinetic binding constants. The experimental procedures, which had been inspired by enzyme-linked immunosorbent assays (ELISA), were validated using ligands of different affinity to carbonic anhydrase II and IX.

Quantitative Assessment of Affinity Selection Performance by Using DNA-Encoded Chemical Libraries.

DNA-encoded chemical libraries are often used for the discovery of ligands against protein targets of interest. These large collections of DNA-barcoded chemical compounds are typically screened by using affinity capture methodologies followed by PCR amplification and DNA sequencing procedures. However, the performance of individual steps in the selection procedures has been scarcely investigated, so far. Herein, the quantitative analysis of selection experiments, by using three ligands with different affinity to carbonic anhydrase IX as model compounds, is described. In the first set of experiments, quantitative PCR (qPCR) procedures are used to evaluate the recovery and selectivity for affinity capture procedures performed on different solid-phase supports, which are commonly used for library screening. In the second step, both qPCR and analysis of DNA sequencing results are used to assess the recovery and selectivity of individual carbonic anhydrase IX ligands in a library, containing 360 000 compounds. Collectively, this study reveals that selection procedures can be efficient for ligands with sub-micromolar dissociation constants to the target protein of interest, but also that selection performance dramatically drops if 104 copies per library member are used as the input.

Site-Specific Labeling of MicroRNA Precursors: A Structure-Activity Relationship Study.

Functionalized oligoribonucleotides are essential tools in RNA chemical biology. Various synthetic routes have been developed over recent years to conjugate functional groups to oligoribonucleotides. However, the presence of the functional group on the oligoribonucleotide backbone can lead to partial or total loss of biological function. The limited knowledge concerning the positioning of functional groups therefore represents a hurdle for the development of oligoribonucleotide chemical tools. Here we describe a systematic investigation of site-specific labeling of pre-miRNAs to identify positions for the incorporation of functional groups, in order not to hinder their processing into active mature miRNAs.

Synthesis of triazolo-fused benzoxazepines and benzoxazepinones via Passerini reactions followed by 1,3-dipolar cycloadditions.

Azidobenzaldehydes can be used in Passerini three-component condensations to synthesize small collections of triazolo-fused heterocycles in an efficient and combinatorial fashion upon post-condensation azide-alkyne cycloadditions. Triazolo-fused benzoxazepinones were obtained in moderate to good overall yields with a concise two-step protocol. Triazolo-fused benzoxazepines were instead prepared by means of a longer, yet straightforward route comprising a Passerini reaction, hydrolysis of the ester moiety, O-alkylation with propargylic bromides, and 1,3-dipolar cycloaddition.

Selective Fragments for the CREBBP Bromodomain Identified from an Encoded Self-assembly Chemical Library.

DNA-encoded chemical libraries (DECLs) are collections of chemical moieties individually coupled to distinctive DNA barcodes. Compounds can be displayed either at the end of a single DNA strand (i. e., single-pharmacophore libraries) or at the extremities of two complementary DNA strands (i. e., dual-pharmacophore libraries). In this work, we describe the use of a dual-pharmacophore encoded self-assembly chemical (ESAC) library for the affinity maturation of a known 4,5-dihydrobenzodiazepinone ring (THBD) acetyl-lysine (KAc) mimic for the cyclic-AMP response element binding protein (CREB) binding protein (CREBBP or CBP) bromodomain. The new pair of fragments discovered from library selection showed a sub-micromolar affinity for the CREBBP bromodomain in fluorescence polarization and ELISA assays, and selectivity against BRD4(1).

Site-Specific Fluorophore Labeling of Guanosines in RNA G-Quadruplexes.

RNA G-quadruplexes are RNA secondary structures that are implicated in many cellular processes. Although conventional biophysical techniques are widely used for their in vitro characterization, more advanced methods are needed to study complex equilibria and the kinetics of their folding. We have developed a new Förster resonance energy-transfer-based method to detect the folding of RNA G-quadruplexes, which is enabled by labeling the 2'-positions of participating guanosines with fluorophores. Importantly, this does not interfere with the required anti conformation of the nucleobase in a quadruplex with parallel topology. Sequential click reactions on the solid phase and in solution using a stop-and-go strategy circumvented the issue of unselective cross-labeling. We exemplified the method on a series of sequences under different assay conditions. In contrast to the commonly used end-labeling approach, our internal labeling strategy would also allow the study of G-quadruplex formation in long functional RNAs.

Impact of a Central Scaffold on the Binding Affinity of Fragment Pairs Isolated from DNA-Encoded Self-Assembling Chemical Libraries.

The screening of encoded self-assembling chemical libraries allows the identification of fragment pairs that bind to adjacent pockets on target proteins of interest. For practical applications, it is necessary to link these ligand pairs into discrete organic molecules, devoid of any nucleic acid component. Here we describe the discovery of a synergistic binding pair for acid alpha-1 glycoprotein and a chemical strategy for the identification of optimal linkers, connecting the two fragments. The procedure yielded a set of small organic ligands, the best of which exhibited a dissociation constant of 9.9 nm, as measured in solution by fluorescence polarization.

OPHA (oxidation-Passerini-hydrolysis-alkylation) strategy: a four-step, one-pot improvement of the alkylative Passerini reaction.

Multicomponent reactions are often recognized for their efficiency and convergency, if compared with multistep organic synthesis. Nevertheless, we here demonstrate that a four-step-one-pot approach (named OPHA strategy for the initials of the four steps involved) is not only able to afford compounds that could not be obtained by an alkylative Passerini reaction but also capable of generating them with minimal loss of atoms and high operational simplicity, as in a typical multicomponent approach.