Identification and Validation of New Interleukin-2 Ligands Using DNA-Encoded Libraries.

Interleukin-2 (IL2) is a pro-inflammatory cytokine that plays a crucial role in immunity, which is increasingly being used for therapeutic applications. There is growing interest in developing IL2-based therapeutics which do not interact with the alpha subunit of the IL2 receptor (CD25) as this protein is primarily found on immunosuppressive regulatory T cells (Tregs). Screenings of a new DNA-encoded library, comprising 669,240 members, provided a novel series of IL2 ligands, subsequently optimized by medicinal chemistry. One of these molecules (compound 18) bound to IL2 with a dissociation constant of 0.34 µM was able to form a kinetically stable complex with IL2 in size-exclusion chromatography and recognized the CD25-binding site as evidenced by competition experiments with the NARA1 antibody. Compound 18 and other members of the series may represent the starting point for the discovery of potent small-molecule modulators of IL2 activity, abrogating the binding to CD25.

DNA-Encoded Chemical Libraries: A Comprehensive Review with Succesful Stories and Future Challenges.

DNA-encoded chemical libraries (DELs) represent a versatile and powerful technology platform for the discovery of small-molecule ligands to protein targets of biological and pharmaceutical interest. DELs are collections of molecules, individually coupled to distinctive DNA tags serving as amplifiable identification barcodes. Thanks to advances in DNA-compatible reactions, selection methodologies, next-generation sequencing, and data analysis, DEL technology allows the construction and screening of libraries of unprecedented size, which has led to the discovery of highly potent ligands, some of which have progressed to clinical trials. In this Review, we present an overview of diverse approaches for the generation and screening of DEL molecular repertoires. Recent success stories are described, detailing how novel ligands were isolated from DEL screening campaigns and were further optimized by medicinal chemistry. The goal of the Review is to capture some of the most recent developments in the field, while also elaborating on future challenges to further improve DEL technology as a therapeutic discovery platform.

An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications.

We describe the development of OncoFAP, an ultra-high-affinity ligand of fibroblast activation protein (FAP) for targeting applications with pan-tumoral potential. OncoFAP binds to human FAP with affinity in the subnanomolar concentration range and cross-reacts with the murine isoform of the protein. We generated various fluorescent and radiolabeled derivatives of OncoFAP in order to study biodistribution properties and tumor-targeting performance in preclinical models. Fluorescent derivatives selectively localized in FAP-positive tumors implanted in nude mice with a rapid and homogeneous penetration within the neoplastic tissue. Quantitative in vivo biodistribution studies with a lutetium-177-labeled derivative of OncoFAP revealed a preferential localization in tumors at doses of up to 1,000 nmol/kg. More than 30% of the injected dose had already accumulated in 1 g of tumor 10 min after intravenous injection and persisted for at least 3 h with excellent tumor-to-organ ratios. OncoFAP also served as a modular component for the generation of nonradioactive therapeutic products. A fluorescein conjugate mediated a potent and FAP-dependent tumor cell killing activity in combination with chimeric antigen receptor (CAR) T cells specific to fluorescein. Similarly, a conjugate of OncoFAP with the monomethyl auristatin E-based Vedotin payload was well tolerated and cured tumor-bearing mice in combination with a clinical-stage antibody-interleukin-2 fusion. Collectively, these data support the development of OncoFAP-based products for tumor-targeting applications in patients with cancer.

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.

Critical Evaluation of Photo-cross-linking Parameters for the Implementation of Efficient DNA-Encoded Chemical Library Selections.

The growing importance of DNA-encoded chemical libraries (DECLs) as tools for the discovery of protein binders has sparked an interest for the development of efficient screening methodologies, capable of discriminating between high- and medium-affinity ligands. Here, we present a systematic investigation of selection methodologies, featuring a library displayed on single-stranded DNA, which could be hybridized to a complementary oligonucleotide carrying a diazirine photoreactive group. Model experiments, performed using ligands of different affinity to carbonic anhydrase IX, revealed a recovery of preferential binders up to 10%, which was mainly limited by the highly reactive nature of carbene intermediates generated during the photo-cross-linking process. Ligands featuring acetazolamide or p-phenylsulfonamide exhibited a higher recovery compared to their counterparts based on 3-sulfamoyl benzoic acid, which had a lower affinity toward the target. A systematic evaluation of experimental parameters revealed conditions that were ideally suited for library screening, which were used for the screening of a combinatorial DECL library, featuring 669 240 combinations of two sets of building blocks. Compared to conventional affinity capture procedures on protein immobilized on solid supports, photo-cross-linking provided a better discrimination of low-affinity CAIX ligands over the background signal and therefore can be used as a tandem methodology with the affinity capture procedures.

DNA-Compatible Diazo-Transfer Reaction in Aqueous Media Suitable for DNA-Encoded Chemical Library Synthesis.

DNA-encoded chemical libraries (DECLs) are increasingly employed in hit discovery toward proteins of pharmaceutical interest. Protected amino acids are the most commonly used building blocks for the construction of DECLs; therefore, the expansion of reaction scope with the subsequent free amine is highly desired. Here, we developed a robust DNA-compatible diazo-transfer reaction using imidazole-1-sulfonyl azide tetrafluoroborate salt converting a wide range of primary amines into their corresponding azides in good to excellent yields.

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.

Optimized Reaction Conditions for Amide Bond Formation in DNA-Encoded Combinatorial Libraries.

DNA-encoded combinatorial libraries are increasingly being used as tools for the discovery of small organic binding molecules to proteins of biological or pharmaceutical interest. In the majority of cases, synthetic procedures for the formation of DNA-encoded combinatorial libraries incorporate at least one step of amide bond formation between amino-modified DNA and a carboxylic acid. We investigated reaction conditions and established a methodology by using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, 1-hydroxy-7-azabenzotriazole and N,N'-diisopropylethylamine (EDC/HOAt/DIPEA) in combination, which provided conversions greater than 75% for 423/543 (78%) of the carboxylic acids tested. These reaction conditions were efficient with a variety of primary and secondary amines, as well as with various types of amino-modified oligonucleotides. The reaction conditions, which also worked efficiently over a broad range of DNA concentrations and reaction scales, should facilitate the synthesis of novel DNA-encoded combinatorial libraries.

Tankyrase 1 Inhibitors with Drug-like Properties Identified by Screening a DNA-Encoded Chemical Library.

We describe the synthesis and screening of a DNA-encoded chemical library containing 76230 compounds. In this library, sets of amines and carboxylic acids are directly linked producing encoded compounds with compact structures and drug-like properties. Affinity screening of this library yielded inhibitors of the potential pharmaceutical target tankyrase 1, a poly(ADP-ribose) polymerase. These compounds have drug-like characteristics, and the most potent hit compound (X066/Y469) inhibited tankyrase 1 with an IC50 value of 250 nM.

"Cap-and-Catch" Purification for Enhancing the Quality of Libraries of DNA Conjugates.

The potential of DNA-encoded combinatorial libraries (DECLs) as tools for hit discovery crucially relies on the availability of methods for their synthesis at acceptable purity and quality. Incomplete reactions in the presence of DNA can noticeably affect the purity of DECLs and methods to selectively remove unreacted oligonucleotide-based starting products would likely enhance the quality of DECL screening results. We describe an approach to selectively remove unreacted oligonucleotide starting products from reaction mixtures and demonstrate its applicability in the context of acylation of amino-modified DNA. Following an amide bond forming reaction, we treat unreacted amino-modified DNAs with biotinylating reagents and isolate the corresponding biotinylated oligonucleotides from the reaction mixture by affinity capture on streptavidin-coated sepharose. This approach, which yields the desired DNA-conjugate at enhanced purity, can be applied both to reactions performed in solution and to procedures in which DNA is immobilized on an anion exchange solid support.

Small targeted cytotoxics from DNA-encoded chemical libraries.

Conventional chemotherapeutic drugs do not selectively localize to tumors, causing undesired toxicities to healthy organs, and precluding the escalation to therapeutically active regimens. The selective delivery at sites of disease of potent effector molecules represents a promising strategy for the treatment of cancer and other diseases. High affinity antibodies towards disease-associated antigens are currently the vehicles of choice for the targeted delivery of payloads. Low molecular weight ligands have the potential to overcome some of the intrinsic limitations associated with antibodies, and have recently been proposed for the development of a novel class of targeted therapeutics. However, the identification of binding molecules, which display high affinity properties and exquisite specificity against protein of therapeutic interest, remains a great challenge. DNA-encoded chemical library technology relies on small molecule libraries of unprecedented size to identify high affinity ligands towards specific target proteins, and could help in the development of next generation targeted cytotoxics.

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.

Supramolecular polymerization of oligopyrenotides--control by single, natural nucleotides.

Amphiphilic heptapyrenotides (Py(7)) assemble into supramolecular polymers. Here we present a comprehensive spectroscopic study of aggregates and co-aggregates of the non-chiral Py(7) and its mono- or di-substituted nucleotide analogs (Py(7)-N and N-Py(7)-N'). The data show that the formation of supramolecular polymers from oligopyrenotides is highly sensitive to the nature of the attached, chiral auxiliary. A single natural nucleotide may be sufficient for the fine tuning of the aggregates' properties by changing the mechanism of aggregation from an isodesmic to a nucleation-elongation process, which results in a high degree of amplification of chirality in the formed supramolecular polymers. Watson-Crick complementarity does not play a significant role, since co-aggregates of oligomers modified with complementary nucleotides show no signs of supramolecular polymerization. Depending on the nucleotide, the helical sense of the polymers is shifted to an M-helix or a P-helix. The findings demonstrate the value of oligopyrenotides as oligomeric building blocks for the generation of optically active supramolecular polymers.

Fluorescent DNA chemosensors: identification of bacterial species by their volatile metabolites.

Polyfluorophores built on a DNA scaffold (ODFs) were synthesized and tested for fluorescence responses to the volatiles from M. tuberculosis, E. coli and P. putida in closed Petri dishes. Two sensors in a pattern-based response could distinguish the bacterial strains accurately, suggesting the use of ODFs in rapid identification of infectious agents.

Differentiating a diverse range of volatile organic compounds with polyfluorophore sensors built on a DNA scaffold.

Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which DNA bases are replaced with fluorophores. A preliminary study reported that some sequences of ODFs were able to respond to a few organic small molecules in the vapor phase, giving a change in fluorescence. Here, we follow up on this finding by investigating a larger range of volatile organic analytes, and a considerably larger set of sensors. A library of tetramer ODFs of 2401 different sequences was prepared by using combinatorial methods, and was screened in air for fluorescence responses to a set of ten different volatile organics, including multiple aromatic and aliphatic compounds, acids and bases, varied functional groups, and closely related structures. Nineteen responding sensors were selected and characterized. These sensors were cross-screened against all ten analytes, and responses were measured qualitatively (by changes in color and intensity) and quantitatively (by measuring ΔR, ΔG, and ΔB values averaged over five to six sensor beads; R=red, G=green, B=blue). The results show that sensor responses were diverse, with a single sensor responding differently to as many as eight of the ten analytes; multiple classes of responses were seen, including quenching, lighting-up, and varied shifts in wavelength. Responses were strong, with raw ΔR, ΔG, and ΔB values of as high as >200 on a 256-unit scale and unamplified changes in many cases apparent to the naked eye. Sensors were identified that could distinguish clearly between even very closely related compounds such as acrolein and acrylonitrile. Statistical methods were applied to select a small set of four sensors that, as a pattern response, could distinguish between all ten analytes with high confidence. Sequence analysis of the full set of sensors suggested that sequence/order of the monomer components, and not merely composition, was highly important in the responses.

Binding of Europium(III) to a non-nucleosidic phenanthroline linker in DNA.

Eu(III) complexes of DNA containing a non-nucleosidic linker, a derivative of 1,10-phenanthroline-2,6-dicarboxamide (Q), are studied with the goal of forming novel lanthanide ion binding sites that are incorporated in the backbone of DNA. One oligonucleotide is short and unstructured (TTTQTTT (QT6)) and the other (5'-AGCTCGGTCAQCGAGAGTGCA-3' (SQ)) is studied both in single-stranded form and in the presence of a partially complementary DNA strand. Luminescence spectroscopy studies show that Eu(III) binds to SQ, QT6, AQB, or QB 1100-, 56-, 23-, or 27-fold more tightly, respectively, than to a simple 1,10-phenanthroline-2,6-dicarboxamide ligand (Q1). Direct excitation and phenanthroline sensitized luminescence spectroscopy supports binding of Eu(III) to the phenanthroline linker in QT6 and in double-stranded DNA formed from SQ and partially complementary sequences that place Q in a bulge-like position. Eu(III) hydration numbers range from 3 to 5 when bound to the phenanthroline moiety in modified DNA, consistent with binding to the tetradentate linker and, in some cases, coordination to other groups in the DNA. Thermal melting experiments show that Q in a bulge-like structure stabilizes double-stranded DNA and that Eu(III) binding does not markedly affect the stability of the duplex.