Asymmetry of Dual-Display DNA-Encoded Chemical Libraries.

While dual-display DNA-encoded chemical libraries (DELs) are increasingly employed for ligand discovery, some of their fundamental properties have not yet been studied in-depth. Aided with fluorescence polarization experiments, we demonstrate that dual-display DELs are intrinsically asymmetrical entities, and we deduce practical guidelines to perform better-informed on-DNA hit validation from these libraries.

Site-Specific Chemical Modification of a Cytokine Mimic for Small Molecule-Based Tumor Targeting.

The targeted delivery of bioactive proteins, such as cytokines, for cancer immunotherapy approaches mostly relies on antibodies or antibody fragments. However, fusion proteins may display low tissue penetration due to a large molecular size. Small molecule ligands with high affinity toward tumor-associated antigens provide a promising alternative for the selective delivery of cytokines to tumor lesions. We developed a one-pot procedure for the site-specific thiazolidine formation between an aldehyde bearing small molecule and the in situ generated N-terminal cysteine of a bioactive protein. Thereby, neoleukin-2/15 (Neo-2/15), a computationally engineered interleukin-2 and -15 mimic, was chemically conjugated to acetazolamide plus, a potent carbonic anhydrase IX (CAIX) ligand. The conjugate retained the biological activity of Neo-2/15 and revealed its ability to accumulate in renal cell carcinoma (SK-RC-52) xenografts upon systemic intravenous administration. The results highlight the potential of small molecule targeting moieties to drive the accumulation of a protein cargo to the respective disease site while conserving the small construct size.

RGD Cyclopeptide Equipped with a Lysine-Engaging Salicylaldehyde Showing Enhanced Integrin Affinity and Cell Detachment Potency.

Salicylaldehyde (SA) derivatives are emerging as useful fragments to obtain reversible-covalent inhibitors interacting with the lysine residues of the target protein. Here the SA installation at the C terminus of an integrin-binding cyclopeptide, leading to enhanced ligand affinity for the receptor as well as stronger biological activity in cultured glioblastoma cells is reported.

Impact of DNA-Encoded Chemical Library Technology on Drug Discovery.

DNA-Encoded Chemical Libraries (DELs) have gained momentum over the recent years for the discovery of small-molecule ligands and the technology has been integrated in most of the larger pharmaceutical companies. With this perspective we would like to summarize the development of DEL technology and present some representative DEL-derived hits which may soon enter the pharmaceutical market.

A DNA-Encoded Chemical Library Based on Peptide Macrocycles.

The synthesis and characterization of a novel DNA-encoded library of macrocyclic peptide derivatives are described; the macrocycles are based on three sets of proteinogenic and non-proteinogenic amino acid building blocks and featuring the use of copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction for ring closure. The library (termed YO-DEL) which contains 1 254 838 compounds, was encoded with DNA in single-stranded format and was screened against target proteins of interest using affinity capture procedures and photocrosslinking. YO-DEL selections yielded specific binders against serum albumins, carbonic anhydrases and NKp46, a marker of activated Natural Killer cells.

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.

Large screening of DNA-compatible reaction conditions for Suzuki and Sonogashira cross-coupling reactions and for reverse amide bond formation.

Progress in DNA-encoded chemical library synthesis and screening crucially relies on the availability of DNA-compatible reactions, which proceed with high yields and excellent purity for a large number of possible building blocks. In the past, experimental conditions have been presented for the execution of Suzuki and Sonogashira cross-coupling reactions on-DNA. In this article, our aim was to optimize Suzuki and Sonogashira reactions, comparing our results to previously published procedures. We have tested the performance of improved conditions using 606 building blocks (including boronic acids, pinacol boranes and terminal alkynes), achieving >70% conversion for 84% of the tested molecules. Moreover, we describe efficient experimental conditions for the on-DNA synthesis of amide bonds, starting from DNA derivatives carrying a carboxylic acid moiety and 300 primary, secondary and aromatic amines, as amide bonds are frequently found in DNA-encoded chemical libraries thanks to their excellent DNA compatibility.

Complexation with a Cognate Antibody Fragment Facilitates Affinity Measurements of Fluorescein-Linked Small Molecule Ligands.

The availability of reliable methods for the characterization of the binding of small molecule ligands to protein targets is crucially important for drug discovery. We have adapted a method, routinely used for the characterization of monoclonal antibodies (enzyme-linked immunosorbent assay, or "ELISA"), to small molecule ligands, using fluorescein conjugates and antifluorescein antibodies as detection reagents. The new small molecule-ELISA methodology was tested using a panel of binders specific to carbonic anhydrase II, with dissociation constants ranging between 6 µM and 14 nM. An excellent agreement was found between ELISA measurements and fluorescence polarization results. The methodology was also extended to BIAcore measurements and implemented for ligands coupled to oligonucleotides. Small molecule-ELISA procedures are particularly useful in the context of DNA-encoded libraries, for which hit validation procedures need to be performed on dozens of candidate molecules and hit compounds can be conveniently resynthesized on DNA.

Comparative evaluation of DNA-encoded chemical selections performed using DNA in single-stranded or double-stranded format.

DNA-encoded chemical libraries (DEL) are increasingly being used for the discovery and optimization of small organic ligands to proteins of biological or pharmaceutical interest. The DNA fragments, that serve as amplifiable identification barcodes for individual compounds in the library, are typically used in double-stranded DNA format. To the best of our knowledge, a direct comparison of DEL selections featuring DNA in either single- or double-stranded DNA format has not yet been reported. In this article, we describe a comparative evaluation of selections with two DEL libraries (named GB-DEL and NF-DEL), based on different chemical designs and produced in both single- and double-stranded DNA format. The libraries were selected in identical conditions against multiple protein targets, revealing comparable and reproducible fingerprints for both types of DNA formats. Surprisingly, selections performed with single-stranded DNA barcodes exhibited improved enrichment factors compared to double-stranded DNA. Using high-affinity ligands to carbonic anhydrase IX as benchmarks for selection performance, we observed an improved selectivity for the NF-DEL library (on average 2-fold higher enrichment factors) in favor of single-stranded DNA. The enrichment factors were even higher for the GB-DEL selections (approximately 5-fold), compared to the same library in double-stranded DNA format. Collectively, these results indicate that DEL libraries can conveniently be synthesized and screened in both single- and double-stranded DNA format, but single-stranded DNA barcodes typically yield enhanced enrichment factors.

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.

Automated and enhanced extraction of a small molecule-drug conjugate using an enzyme-inhibitor interaction based SPME tool followed by direct analysis by ESI-MS.

We report a novel, fast, and automatic SPME-based method capable of extracting a small molecule-drug conjugate (SMDC) from biological matrices. Our method relies on the extraction of the drug conjugate followed by direct elution into an electrospray mass spectrometer (ESI-MS) source for qualitative and quantitative analysis. We designed a tool for extracting the targeting head of a recently synthesized SMDC, which includes acetazolamide (AAZ) as high-affinity ligand specific to carbonic anhydrase IX. Specificity of the extraction was achieved through systematic optimization. The design of the extraction tool is based on noncovalent and reversible interaction between AAZ and CAII that is immobilized on the SPME extraction phase. Using this approach, we showed a 330% rise in extracted AAZ signal intensity compared to a control, which was performed in the absence of CAII. A linear dynamic range from 1.2 to 25 µg/ml was found. The limits of detection (LOD) of extracted AAZ from phosphate-buffered saline (PBS) and human plasma were 0.4 and 1.2 µg/ml, respectively. This with a relative standard deviation of less than 14% (n = 40) covers the therapeutic range. Graphical abstract.

Affinity Enhancement of Protein Ligands by Reversible Covalent Modification of Neighboring Lysine Residues.

The discovery of protein ligands, capable of forming a reversible covalent bond with amino acid residues on a protein target of interest, may represent a general strategy for the discovery of potent small-molecule inhibitors. We analyzed the ability of different aromatic aldehydes to form imines by reaction with lysine using 1 H NMR techniques. 2-Hydroxybenzaldehyde derivatives were found to efficiently form imines in the millimolar concentration range. These benzaldehyde derivatives could increase the binding affinity of protein ligands towards the cognate protein target. Affinity maturation was achieved not only by displaying ligand and aldehyde moieties on two complementary locked nucleic acid strands but also by incorporating the binding fragments in a single small-molecule ligand. The affinity gain was only observed when lysine residues were accessible in the immediate surroundings of the ligand-binding site and could be abrogated by quenching with a molar excess of hydroxylamine.

Quantitative PCR is a Valuable Tool to Monitor the Performance of DNA-Encoded Chemical Library Selections.

Phage-display libraries and DNA-encoded chemical libraries (DECLs) represent useful tools for the isolation of specific binding molecules from large combinatorial sets of compounds. With both methods, specific binders are recovered at the end of affinity capture procedures by using target proteins of interest immobilized on a solid support. However, although the efficiency of phage-display selections is routinely quantified by counting the phage titer before and after the affinity capture step, no similar quantification procedures have been reported for the characterization of DECL selections. In this article, we describe the potential and limitations of quantitative PCR (qPCR) methods for the evaluation of selection efficiency by using a combinatorial chemical library with more than 35 million compounds. In the experimental conditions chosen for the selections, a quantification of DNA input/recovery over five orders of magnitude could be performed, revealing a successful enrichment of abundant binders, which could be confirmed by DNA sequencing. qPCR provided rapid information about the performance of selections, thus facilitating the optimization of experimental conditions.

Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries.

DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article we present new methods based on hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides; these facilitate the determination of affinity constants and kinetic dissociation constants. The experimental procedures were demonstrated with acetazolamide, a binder to carbonic anhydrase IX with a dissociation constant in the nanomolar range. The detection of binding events was compatible not only with fluorescence polarization methodologies, but also with Alphascreen technology and with microscale thermophoresis.

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

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.

DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries.

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

Identification of structure-activity relationships from screening a structurally compact DNA-encoded chemical library.

Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA-encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL-100K, a DNA-encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA-sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate-specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small-molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding.

Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.

DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

Highly pure DNA-encoded chemical libraries by dual-linker solid-phase synthesis.

The first drugs discovered using DNA-encoded chemical library (DEL) screens have entered late-stage clinical development. However, DEL technology as a whole still suffers from poor chemical purity resulting in suboptimal performance. In this work, we report a technique to overcome this issue through self-purifying release of the DEL after magnetic bead-based synthesis. Both the first and last building blocks of each assembled library member were linked to the beads by tethers that could be cleaved by mutually orthogonal chemistry. Sequential cleavage of the first and last tether, with washing in between, ensured that the final library comprises only the fully complete compounds. The outstanding purity attained by this approach enables a direct correlation of chemical display and encoding, allows for an increased chemical reaction scope, and facilitates the use of more diversity elements while achieving greatly improved signal-to-noise ratios in selections.