Discovery of Small‐Molecule Interleukin‐2 Inhibitors from a DNA‐Encoded Chemical Library

Libraries of chemical compounds individually coupled to encoding DNA tags (DNA‐encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high‐quality DNA‐encoded chemical library comprising 30 000 drug‐like compounds; this was screened in 170 different affinity capture experiments. High‐throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor‐associated antigen carbonic anhydrase IX (CA IX) and the pro‐inflammatory cytokine interleukin‐2 (IL‐2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL‐2 was confirmed by molecular docking. Our findings suggest that DNA‐encoded chemical libraries allow the facile identification of drug‐like ligands principally to any protein of choice, including molecules capable of disrupting high‐affinity protein–protein interactions.     

DNA‐Encoded Dynamic Combinatorial Chemical Libraries

Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA‐encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal‐to‐noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA‐encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high‐affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA‐templated chemical reactions.     

Functionality‐Independent DNA Encoding of Complex Natural Products

DNA encoded chemical libraries (DELs) link the powers of genetics and chemical synthesis via combinatorial optimization. Through combinatorial chemistry, DELs can grow to the unprecedented size of billions to trillions. To take full advantage of the DEL approach, linking the power of genetics directly to chemical structures would offer even greater diversity in a finite chemical world. Natural products have evolved an incredible structural diversity along with their biological evolution. Herein, we used traditional Chinese medicines (TCMs) as examples in a late‐stage modification toolbox approach to annotate these complex organic compounds with amplifiable DNA barcodes, which could be easily incorporated into a DEL. The method of end‐products labeling also generates a cluster of isomers with a single DNA tag at different sites. These isomers provide an additional spatial diversity for multiple accessible pockets of targeted proteins. Notably, a novel PARP1 inhibitor from TCM has been identified from the natural products enriched DEL (nDEL).     

A DNA‐Encoded Chemical Library Incorporating Elements of Natural Macrocycles

Here we show a seven‐step chemical synthesis of a DNA‐encoded macrocycle library (DEML) on DNA. Inspired by polyketide and mixed peptide‐polyketide natural products, the library was designed to incorporate rich backbone diversity. Achieving this diversity, however, comes at the cost of the custom synthesis of bifunctional building block libraries. This study outlines the importance of careful retrosynthetic design in DNA‐encoded libraries, while revealing areas where new DNA synthetic methods are needed.     

Screening of Three Transition Metal‐Mediated Reactions Compatible with DNA‐Encoded Chemical Libraries

The construction of DNA‐encoded chemical libraries (DECLs) crucially relies on the availability of chemical reactions, which are DNA‐compatible and which exhibit high conversion rates for a large number of diverse substrates. In this work, we present our optimization and validation procedures for three copper and palladium‐catalyzed reactions (Suzuki cross‐coupling, Sonogashira cross‐coupling, and copper(I)‐catalyzed alkyne‐azide cycloaddition (CuAAC)), which have been successfully used by our group for the construction of large encoded libraries.     

Small Targeted Cytotoxics: Current State and Promises from DNA‐Encoded Chemical Libraries

The targeted delivery of potent cytotoxic agents has emerged as a promising strategy for the treatment of cancer and other serious conditions. Traditionally, antibodies against markers of disease have been used as drug‐delivery vehicles. More recently, lower molecular weight ligands have been proposed for the generation of a novel class of targeted cytotoxics with improved properties. Advances in this field crucially rely on efficient methods for the identification and optimization of organic molecules capable of high‐affinity binding and selective recognition of target proteins. The advent of DNA‐encoded chemical libraries allows the construction and screening of compound collections of unprecedented size. In this Review, we survey developments in the field of small ligand‐based targeted cytotoxics and show how innovative library technologies will help develop the drugs of the future.     

Selection of DNA‐Encoded Small Molecule Libraries Against Unmodified and Non‐Immobilized Protein Targets

The selection of DNA‐encoded libraries against biological targets has become an important discovery method in chemical biology and drug discovery, but the requirement of modified and immobilized targets remains a significant disadvantage. With a terminal protection strategy and ligand‐induced photo‐crosslinking, we show that iterated selections of DNA‐encoded libraries can be realized with unmodified and non‐immobilized protein targets.     

Selection of New Sequence‐Selective Unnatural Peptides Binding to Double‐Stranded Deoxyribonucleic Acids (dsDNA) by Means of a Gel‐Retardation Experiment for Library Analysis

Previously, we developed a methodology for the solid‐phase screening of peptide libraries for interaction with double‐stranded deoxyribonucleic acids (dsDNA). In the search for new and more‐potent DNA ligands, we investigated the strategy of solution‐phase screening of chemical libraries consisting of unnatural oligopeptides. After synthesis of the selected amino acid building blocks, libraries were constructed with the general structure Ac‐Arg‐Ual‐Sar‐X¹‐X²‐X³‐Arg‐NH₂, where X represents each of twelve unnatural or natural amino acids. Optimization of the sequence of binding peptides was performed with an iterative deconvolution procedure. Selection of interacting peptides was carried out in solution by means of gel‐retardation experiments, starting with libraries of 144 compounds. A 14‐base‐pair double‐stranded DNA fragment was chosen as the target. After several cycles of synthesis and screening of libraries and individual peptides, an oligopeptide was selected with an apparent dissociation constant of 9⋅10⁻⁵ M , as determined by gel‐retardation experiments. This peptide was studied by NMR spectroscopy. A certain degree of conformational pre‐organization of the peptides was shown by temperature‐dependent circular‐dichroism experiments. Finally, DNase‐I‐footprinting studies indicated a preferential interaction with a 6‐base‐pair mixed sequence 5′‐CTGCAT‐3′. This study demonstrates that gel‐shift experiments can be used for the solution‐phase screening of library mixtures of peptides against dsDNA. In general, this technique allows the selection of new sequence‐selective dsDNA‐interacting molecules. Furthermore, novel dsDNA‐binding unnatural oligopeptides were developed with affinities in the 0.1 mM range.     

Towards the Development of Structure‐Selective G‐Quadruplex‐Binding Indolo[3,2‐b]quinolines

The interaction of phenyl‐substituted indolo[3,2‐b]quinolines with DNA G‐quadruplexes of different topology were studied by using a combination of spectroscopic and calorimetric methodologies. N5‐Methylated indoloquinoline derivatives (^MePIQ) with an aminoalkyl side chain exhibit high affinities for the parallel‐stranded MYC quadruplex and a (3+1)‐hybrid structure combined with an excellent discrimination against the antiparallel thrombin‐binding aptamer (TBA) and the human telomeric (HT) quadruplexes. Dissociation constants for the binding of the ligand to the MYC quadruplex are in the submicromolar range, being below the corresponding dissociation constants for the antiparallel‐stranded quadruplexes by about one order of magnitude. Competition experiments with double‐helical DNA reveal the impact of indoloquinoline structural features on the selectivity for the parallel quadruplex relative to duplex DNA. Based on a calorimetric analysis binding to MYC is shown to be equally driven by favorable enthalpic and entropic contributions with no significant impact on the type of cation present.     

20 years of DNA-encoded chemical libraries

The identification of specific binding molecules is a central problem in chemistry, biology and medicine. Therefore, technologies, which facilitate ligand discovery, may substantially contribute to a better understanding of biological processes and to drug discovery. DNA-encoded chemical libraries represent a new inexpensive tool for the fast and efficient identification of ligands to target proteins of choice. Such libraries consist of collections of organic molecules, covalently linked to a unique DNA tag serving as an amplifiable identification bar code. DNA-encoding enables the in vitro selection of ligands by affinity capture at sub-picomolar concentrations on virtually any target protein of interest, in analogy to established selection methodologies like antibody phage display. Multiple strategies have been investigated by several academic and industrial laboratories for the construction of DNA-encoded chemical libraries comprising up to millions of DNA-encoded compounds. The implementation of next generation high-throughput sequencing enabled the rapid identification of binding molecules from DNA-encoded libraries of unprecedented size. This article reviews the development of DNA-encoded library technology and its evolution into a novel drug discovery tool, commenting on challenges, perspectives and opportunities for the different experimental approaches.     

Initial DNA Interactions of the Binuclear Threading Intercalator Λ,Λ‐[μ‐bidppz(bipy)4Ru2]4+: An NMR Study with [d(CGCGAATTCGCG)]2

Binuclear polypyridine ruthenium compounds have been shown to slowly intercalate into DNA, following a fast initial binding on the DNA surface. For these compounds, intercalation requires threading of a bulky substituent, containing one Ru^II, through the DNA base‐pair stack, and the accompanying DNA duplex distortions are much more severe than with intercalation of mononuclear compounds. Structural understanding of the process of intercalation may greatly gain from a characterisation of the initial interactions between binuclear Ru^II compounds and DNA. We report a structural NMR study on the binuclear Ru^II intercalator Λ,Λ‐B (Λ,Λ‐[μ‐bidppz(bipy)₄Ru₂]⁴⁺; bidppz=11,11′‐bis(dipyrido[3,2‐a:2′,3′‐c]phenazinyl, bipy = 2,2′‐bipyridine) mixed with the palindromic DNA [d(CGCGAATTCGCG)]₂. Threading of Λ,Λ‐B depends on the presence and length of AT stretches in the DNA. Therefore, the latter was selected to promote initial binding, but due to the short stretch of AT base pairs, final intercalation is prevented. Structural calculations provide a model for the interaction: Λ,Λ‐B is trapped in a well‐defined surface‐bound state consisting of an eccentric minor‐groove binding. Most of the interaction enthalpy originates from electrostatic and van der Waals contacts, whereas intermolecular hydrogen bonds may help to define a unique position of Λ,Λ‐B. Molecular dynamics simulations show that this minor‐groove binding mode is stable on a nanosecond scale. To the best of our knowledge, this is the first structural study by NMR spectroscopy on a binuclear Ru compound bound to DNA. In the calculated structure, one of the positively charged Ru²⁺ moieties is near the central AATT region; this is favourable in view of potential intercalation as observed by optical methods for DNA with longer AT stretches. Circular dichroism (CD) spectroscopy suggests that a similar binding geometry is formed in mixtures of Λ,Λ‐B with natural calf thymus DNA. The present minor‐groove binding mode is proposed to represent the initial surface interactions of binuclear Ru^II compounds prior to intercalation into AT‐rich DNA.     

Modeling and predicting binding affinity of phencyclidine‐like compounds using machine learning methods

Machine learning methods have always been promising in the science and engineering fields, and the use of these methods in chemistry and drug design has advanced especially since the 1990s. In this study, molecular electrostatic potential (MEP) surfaces of phencyclidine‐like (PCP‐like) compounds are modeled and visualized in order to extract features that are useful in predicting binding affinities. In modeling, the Cartesian coordinates of MEP surface points are mapped onto a spherical self‐organizing map (SSOM). The resulting maps are visualized using electrostatic potential (ESP) values. These values also provide features for a prediction system. Support vector machines and partial least‐squares method are used for predicting binding affinities of compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

Fabrication of Highly Stable Glyco‐Gold Nanoparticles and Development of a Glyco‐Gold Nanoparticle‐Based Oriented Immobilized Antibody Microarray for Lectin (GOAL) Assay

The design of high‐affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco‐gold nanoparticles (glyco‐AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco‐AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70 mol %) and an amphiphilic linker (30 mol %) provide long‐term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco‐AuNPs enable the detection of model plant lectins such as Concanavalin A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop in situ glyco‐AuNPs‐based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of glyconanoparticle‐based biosensors.     

Limiting the Number of Potential Binding Modes by Introducing Symmetry into Ligands: Structure‐Based Design of Inhibitors for Trypsin‐Like Serine Proteases

In the absence of X‐ray data, the exploration of compound binding modes continues to be a challenging task. For structure‐based design, specific features of active sites in different targets play a major role in rationalizing ligand binding characteristics. For example, dibasic compounds have been reported as potent inhibitors of various trypsin‐like serine proteases, the active sites of which contain several binding pockets that can be targeted by cationic moieties. This results in several possible orientations within the active site, complicating the binding mode prediction of such compounds by docking tools. Therefore, we introduced symmetry in bi‐ and tribasic compounds to reduce conformational space in docking calculations and to simplify binding mode selection by limiting the number of possible pocket occupations. Asymmetric bisbenzamidines were used as starting points for a multistage and structure‐guided optimization. A series of 24 final compounds with either two or three benzamidine substructures was ultimately synthesized and evaluated as inhibitors of five serine proteases, leading to potent symmetric inhibitors for the pharmaceutical drug targets matriptase, matriptase‐2, thrombin and factor Xa. This study underlines the relevance of ligand symmetry for chemical biology.     

A Chemistry for Incorporation of Selenium into DNA‐Encoded Libraries

Conventional direct C?H selenylation suffers from simple selenation with limited atom economy and complicated reaction system. In this work, we designed benzoselenazolone as a novel bifunctional selenide reagent for both off‐ and on‐DNA C?H selenylation under rhodium(III) catalysis. We show that using benzoselenazolone allowed production of a series of selenylation products containing an adjacent aminoacyl group in a fast and efficient way, with high atom economy. The synthetic application of this method was demonstrated by taking advantage of the amide functionality as a nucleophile, directing group, and amide coupling partner. This work shows great potential in facilitating rapid construction of selenium‐containing DNA‐encoded chemical libraries (SeDELs), and lays the foundation for the development of selenium‐containing drugs.     

Immunoassays: biological tools for high throughput screening and characterisation of combinatorial libraries

In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the "intelligent" design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.     

The archaeal Sac10b protein family: conserved proteins with divergent functions

Here we review the present state of structural and functional studies of the Sac10b protein family, a class of highly conserved 10 kDa nucleic acid-binding proteins in archaea. Based on biochemical and structural studies, these proteins were originally assigned a role in the structural organization of chromatin; Sac10b proteins of hyperthermophilic archaea, for example, showed tight, unspecific DNA binding. More recently, however, Sac10b proteins of mesophilic archaea were found to interact preferentially with specific DNA sequences thereby affecting the expression of distinct genes. Furthermore, Sac10b proteins of hyperthermophilic, thermophilic and mesophilic archaea were also shown to bind to RNA with distinct affinities and specificities but functional consequences of RNA binding of these proteins, besides perhaps RNA stabilization, have not yet been observed. To better understand the physiological meaning of the various interactions of Sac10b proteins with nucleic acids, future work should concentrate on elucidating the molecular structures of complexes of Sac10b proteins of hyperthermophilic and mesophilic archaea with DNA and RNA. In addition, existing and new X-ray and NMR structures of individual hyperthermophilic Sac10b proteins may represent very good models for introducing thermostability especially in enzymes for industrial use.     

Selection of DNA‐Encoded Dynamic Chemical Libraries for Direct Inhibitor Discovery

Dynamic combinatorial libraries (DCLs) is a powerful tool for ligand discovery in biomedical research; however, the application of DCLs has been hampered by their low diversity. Recently, the concept of DNA encoding has been employed in DCLs to create DNA‐encoded dynamic libraries (DEDLs); however, all current DEDLs are limited to fragment identification, and a challenging process of fragment linking is required after selection. We report an anchor‐directed DEDL approach that can identify full ligand structures from large‐scale DEDLs. This method is also able to convert unbiased libraries into focused ones targeting specific protein classes. We demonstrated this method by selecting DEDLs against five proteins, and novel inhibitors were identified for all targets. Notably, several selective BD1/BD2 inhibitors were identified from the selections against bromodomain 4 (BRD4), an important anti‐cancer drug target. This work may provide a broadly applicable method for inhibitor discovery.     

Synthetic dsDNA‐Binding Peptides Using Natural Compounds as Model

We have developed a series of short DNA‐binding peptides containing newly synthesized, unnatural as well as natural amino acid building blocks. By a combinatorial‐library approach, oligopeptides were developed with moderate dsDNA‐binding affinities. Two strategies were used to further enhance the binding affinity of the lead peptides: Ac‐Arg‐Ual‐Sar‐Chi‐Chi‐Chi‐Arg‐NH₂ and Ac‐Arg‐Cbg‐Cha‐Chi‐Chi‐Tal‐Arg‐NH₂. Site‐selective amino acid substitutions increased the binding affinities up to 2 × 10^?5 M . Further enhancement of the binding affinities could be achieved by coupling of an acridine intercalating unit, using linker arms of different length and flexibility. With the introduction of a new lysine‐based acridine unit, different types of oligopeptide–acridine conjugates were designed using known dsDNA‐binding ligands as model compounds. The binding capacities of these new oligopeptide–acridine conjugates have been investigated by a fluorescent intercalator (ethidium bromide) displacement (FID) assay. With the synthesis of the dipeptide–acridine conjugates, binding affinities in the low micromolar range were obtained (6.4 × 10^?6 M ), which is similar to the binding strength of the well‐known DNA binder Hoechst 33258.