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Background: A safe, effective, and reversible nonhormonal male contraceptive drug is greatly needed for male contraception as well as for circumventing the side effects of female hormonal contraceptives. Phosducin-like 2 (PDCL2) is a testis-specific phosphoprotein in mice and humans. We recently found that male PDCL2 knockout mice are sterile due to globozoospermia caused by impaired sperm head formation, indicating that PDCL2 is a potential target for male contraception. Herein, our study for the first time developed a biophysical assay for PDCL2 allowing us to screen a series of small molecules, to study structure-activity relationships, and to discover two PDCL2 binders with novel chemical structure., Objective: To identify a PDCL2 ligand for therapeutic male contraception, we performed DNA-encoded chemical library (DECL) screening and off-DNA hit validation using a unique affinity selection mass spectrometry (ASMS) biophysical profiling strategy., Materials and Methods: We employed the screening process of DECL, which contains billions of chemically unique DNA-barcoded compounds generated through individual sequences of reactions and different combinations of functionalized building blocks. The structures of the PDCL2 binders are proposed based on the sequencing analysis of the DNA barcode attached to each individual DECL compound. The proposed structure is synthesized through multistep reactions. To confirm and determine binding affinity between the DECL identified molecules and PDCL2, we developed an ASMS assay that incorporates liquid chromatography with tandem mass spectrometry (LC-MS/MS)., Results: After a screening process of PDCL2 with DECLs containing >440 billion compounds, we identified a series of hits. The selected compounds were synthesized as off-DNA small molecules, characterized by spectroscopy data, and subjected to our ASMS/LC-MS/MS binding assay. By this assay, we discovered two novel compounds, which showed good binding affinity for PDCL2 in comparison to other molecules generated in our laboratory and which were further confirmed by a thermal shift assay., Discussion and Conclusion and Relevance: With the ASMS/LC-MS/MS assay developed in this paper, we successfully discovered a PDCL2 ligand that warrants further development as a male contraceptive., (© 2022 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Affinity-based DNA-encoded library (DEL) selection is considered a powerful tool for small molecule drug discovery. Such selections are a multi-round process that involves incubation of a target protein with the DEL, capture of the protein and associated DEL compounds on a solid support, separation of bound molecules from the bulk DEL that is unbound, and recovery of bound DEL molecules. Each step is of great importance in order to achieve successful selections. Here we describe the selection process against a soluble target protein in both the immobilized and in-solution modes., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

G-quadruplexes are four-stranded nucleic acid secondary structures of biological significance and have emerged as an attractive drug target. The G4 formed in the MYC promoter (MycG4) is one of the most studied small-molecule targets, and a model system for parallel structures that are prevalent in promoter DNA G4s and RNA G4s. Molecular docking has become an essential tool in structure-based drug discovery for protein targets, and is also increasingly applied to G4 DNA. However, DNA, and in particular G4, binding sites differ significantly from protein targets. Here we perform the first systematic evaluation of four commonly used docking programs (AutoDock Vina, DOCK 6, Glide, and RxDock) for G4 DNA-ligand binding pose prediction using four small molecules whose complex structures with the MycG4 have been experimentally determined in solution. The results indicate that there are considerable differences in the performance of the docking programs and that DOCK 6 with GB/SA rescoring performs better than the other programs. We found that docking accuracy is mainly limited by the scoring functions. The study shows that current docking programs should be used with caution to predict G4 DNA-small molecule binding modes.

The MYCN gene encodes the transcription factor N-Myc, a driver of neuroblastoma (NB). Targeting G-quadruplexes (G4s) with small molecules is attractive strategy to control the expression of undruggable proteins such as N-Myc. However, selective binders to G4s are challenging to identify due to the structural similarity of many G4s. Here, we report the discovery of a small molecule ligand (4) that targets the noncanonical, hairpin containing G4 structure found in the MYCN gene using small molecule microarrays (SMMs). Unlike many G4 binders, the compound was found to bind to a pocket at the base of the hairpin region of the MYCN G4. This compound stabilizes the G4 and has affinity of 3.5 ± 1.6 μM. Moreover, an improved analog, MY-8, suppressed levels of both MYCN and MYCNOS (a lncRNA embedded within the MYCN gene) in NBEB neuroblastoma cells. This work indicates that the approach of targeting complex, hybrid G4 structures that exist throughout the human genome may be an applicable strategy to achieve selectivity for targeting disease-relevant genes including protein coding (MYCN) as well as non-coding (MYCNOS) gene products., (Published by Oxford University Press on behalf of Nucleic Acids Research 2021.)

Inhibition of hydroxy acid oxidase 1 (HAO1) is a strategy to mitigate the accumulation of toxic oxalate that results from reduced activity of alanine-glyoxylate aminotransferase (AGXT) in primary hyperoxaluria 1 (PH1) patients. DNA-Encoded Chemical Library (DECL) screening provided two novel chemical series of potent HAO1 inhibitors, represented by compounds 3 - 6 . Compound 5 was further optimized via various structure-activity relationship (SAR) exploration methods to 29 , a compound with improved potency and absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) properties. Since carboxylic acid-containing compounds are often poorly permeable and have potential active glucuronide metabolites, we undertook a brief, initial exploration of acid replacements with the aim of identifying non-acid-containing HAO1 inhibitors. Structure-based drug design initiated with Compound 5 led to the identification of a nonacid inhibitor of HAO1, 31 , which has weaker potency and increased permeability.

Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library (DECL) screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function (GOF) mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplar 7 exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant of 7 rapidly generated bispecific nanomolar degraders of ERα, with PROTACs 18 and 21 inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization.

DNA-encoded small molecule libraries (DELs) have facilitated the discovery of novel modulators of many different therapeutic protein targets. We report the first successful screening of a multimillion membered DEL inside a living cell. We demonstrate a novel method using oocytes from the South African clawed frog Xenopus laevis . The large size of the oocytes of 1 μL, or 100 000 times bigger than a normal somatic cell, permits simple injection of DELs, thus resolving the fundamental problem of delivering DELs across cell membranes for in vivo screening. The target protein was expressed in the oocytes fused to a prey protein, to allow specific DNA labeling and hereby discriminate between DEL members binding to the target protein and the endogenous cell proteins. The 194 million member DEL was screened against three pharmaceutically relevant protein targets, p38α, ACSS2, and DOCK5. For all three targets multiple chemical clusters were identified. For p38α, validated hits with single digit nanomolar potencies were obtained. This work demonstrates a powerful new approach to DEL screening, which eliminates the need for highly purified active target protein and which performs the screening under physiological relevant conditions and thus is poised to increase the DEL amenable target space and reduce the attrition rates.

Membrane proteins on the cell surface perform a myriad of biological functions; however, ligand discovery for membrane proteins is highly challenging, because a natural cellular environment is often necessary to maintain protein structure and function. DNA-encoded chemical libraries (DELs) have emerged as a powerful technology for ligand discovery, but they are mainly limited to purified proteins. Here we report a method that can specifically label membrane proteins with a DNA tag, and thereby enable target-specific DEL selections against endogenous membrane proteins on live cells without overexpression or any other genetic manipulation. We demonstrate the generality and performance of this method by screening a 30.42-million-compound DEL against the folate receptor, carbonic anhydrase 12 and the epidermal growth factor receptor on live cells, and identify and validate a series of novel ligands for these targets. Given the high therapeutic significance of membrane proteins and their intractability to traditional high-throughput screening approaches, this method has the potential to facilitate membrane-protein-based drug discovery by harnessing the power of DEL.

Two critical steps in drug development are 1) the discovery of molecules that have the desired effects on a target, and 2) the optimization of such molecules into lead compounds with the required potency and pharmacokinetic properties for translation. DNA-encoded chemical libraries (DECLs) can nowadays yield hits with unprecedented ease, and lead-optimization is becoming the limiting step. Here we integrate DECL screening with structure-based computational methods to streamline the development of lead compounds. The presented workflow consists of enumerating a virtual combinatorial library (VCL) derived from a DECL screening hit and using computational binding prediction to identify molecules with enhanced properties relative to the original DECL hit. As proof-of-concept demonstration, we applied this approach to identify an inhibitor of PARP10 that is more potent and druglike than the original DECL screening hit., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Targeting guanine (G)-rich DNA sequences, folded into non-canonical G-quadruplex (G4) structures, by small ligand molecules is a potential strategy for gene therapy of cancer disease. BRACO-19 has been recently established as a unique (thermodynamically favorable and highly selective) binder, being involved in the external stacking mode of interaction with a G4-DNA formed in the c-Myc oncogene promoter region (P. M. Mitrasinovic, Croat. Chem. Acta 2019, 92, 43-57). Herein, hit-to-lead ligands are identified using high-throughput virtual screening (HTVS). Search of the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases is performed using the key pharmacophore features of BRACO-19. At the very outset, out of a total of 29,009 entries, 95 hits are extracted and evaluated by docking them in the binding sites of G4. Then, 22 hits are chosen by observing the binding free energies. Consequently, 3 hit-to-lead candidates are selected on the basis of structural criteria. Finally, a lead candidate structure is proposed using analog design and considering both the physicochemical requirements for optimal biological activity and a variety of pharmacological standpoints. Implications of the present study for experimental research are discussed.

Transcription factor activator protein-1 (AP-1) binds to cognate DNA and regulates gene expression. In recent decades, small-molecule inhibitors have been developed for therapeutic applications that block AP-1 binding to DNA. However, the mechanism by which small molecules inhibit AP-1-DNA binding remains elusive. Here, computational studies identified a drug-binding site on the AP-1 Fos/Jun apo structure. Induced fit docking of known inhibitors, together with metadynamics simulations to identify the most plausible binding pose, showed a consensus mode of AP-1/inhibitor interaction. The in silico binding mode of the inhibitors suggests a mechanism of AP-1-DNA binding inhibition, where the inhibitors block the base-contacting residues, preclude access of DNA, and prohibit conformational changes of AP-1 upon DNA binding.

FOXO transcription factors are critical regulators of cell homeostasis and steer cell death, differentiation and longevity in mammalian cells. By combined pharmacophore-modeling-based in silico and fluorescence polarization-based screening we identified small molecules that physically interact with the DNA-binding domain (DBD) of FOXO3 and modulate the FOXO3 transcriptional program in human cells. The mode of interaction between compounds and the FOXO3-DBD was assessed via NMR spectroscopy and docking studies. We demonstrate that compounds S9 and its oxalate salt S9OX interfere with FOXO3 target promoter binding, gene transcription and modulate the physiologic program activated by FOXO3 in cancer cells. These small molecules prove the druggability of the FOXO-DBD and provide a structural basis for modulating these important homeostasis regulators in normal and malignant cells., Competing Interests: JH, VO, TK, NK, BR, KP, VD, MA, KK, DS, TA, JV, PO, TO, MA No competing interests declared, (© 2019, Hagenbuchner et al.)

We report the design and synthesis of a DNA-encoded one-bead one-compound library of cyclic peptoids composed of more than 11 million molecules. We show that affinity-based screening of this large library can identify cyclic peptoid ligands for a target protein. In this work, we developed a simple method for amplifying the PCR product from DNA tags on a single bead, thereby enabling determination of the structures of hit cyclic peptoids with no need for high-throughput sequencing and complicated data analysis. We also developed a sublibrary screening strategy to minimize false positives caused by the interference of coding DNA tags before starting laborious and impractical hit confirmation. Given the simplicity and robustness of the synthesis and screening, along with the desirable features of macrocyclic peptoids including improved conformational rigidity, our method will be highly useful for discovering biologically active molecules modulating challenging targets such as protein-protein interactions that are not easily targeted by typical peptidomimetics and small-molecules.

As aberrant activity of protein kinases is observed in many disease states, these enzymes are common targets for therapeutics and detection of activity levels. The development of non-natural protein kinase substrates offers an approach to protein substrate competitive inhibitors, a class of kinase inhibitors with promise for improved specificity. Also, kinase activity detection approaches would benefit from substrates with improved activity and specificity. Here, we apply a substrate-mediated selection to a peptidomimetic DNA-encoded chemical library for enrichment of molecules that can be phosphorylated by the protein tyrosine kinase, c-Src. Several substrates were identified and characterized for activity. A lead compound ( SrcDEL10 ) showed both the ability to serve as a substrate and to promote ATP hydrolysis by the kinase. In inhibition assays, compounds displayed IC 50' s ranging from of 8-100 µM. NMR analysis of SrcDEL10 bound to the c-Src:ATP complex was conducted to characterize the binding mode. An ester derivative of the lead compound demonstrated cellular activity with inhibition of Src-dependent signaling in cell culture. Together, the results show the potential for substrate-mediated selections of DNA-encoded libraries to discover molecules with functions other than simple protein binding and offer a new discovery method for development of synthetic tyrosine kinase substrates.

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)., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The human genome is replete with repetitive DNA sequences that can fold into thermodynamically stable secondary structures such as hairpins and quadruplexes. Cellular enzymes exist to cope with these structures whose stable accumulation would result in DNA damage through interference with DNA transactions such as transcription and replication. Therefore, the chemical stabilization of secondary DNA structures offers an attractive way to foster DNA transaction-associated damages to trigger cell death in proliferating cancer cells. While much emphasis has been recently given to DNA quadruplexes, we focused here on three-way DNA junctions (TWJ) and report on a strategy to identify TWJ-targeting agents through a combination of in vitro techniques (TWJ-screen, polyacrylamide gel electrophoresis, fluorescence resonance energy transfer-melting, electrospray ionization mass spectrometry, dialysis equilibrium, and sulforhodamine B assays). We designed a complete workflow and screened 1200 compounds to identify promising TWJ ligands selected on stringent criteria in terms of TWJ-folding ability, affinity, and selectivity.

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 10 4 copies per library member are used as the input., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA-encoded library technology (ELT) is a cutting-edge enabling technology platform for drug discovery. Here we describe how to design and synthesize a macrocyclic DNA-encoded library; how to perform selection, sequencing, and data analysis to identify potential active peptides; and how to synthesize off-DNA peptides to confirm activity. This approach provides an effective tool for pharmaceutical research based on peptides.

Major problems in cancer chemotherapy are toxicity, resistance, and cancer heterogeneity. A new theranostic paradigm has been proposed by the authors. Many million small molecules (SM) are bound to the proteins extracted from a patient's cancer. SM that also bind proteins extracted from normal human tissues are subtracted from the cancer protein bound SM leaving a large array of SM targeting many sites on each of the cancer biomarkers. Targeting many more than the conventional 1 - 4 cancer biomarkers will reduce development of tumor resistance. After several cycles of selection and counter selection, DNA codes appended to the SM will be PCR amplified to provide templates for restricted libraries of the SM to improve selectivity and sensitivity. The large array of selected and counter selected SM assures that many of the compounds in the array will penetrate the cell membrane and bind to intracellular targets, low tumor resistance, low background for imaging, low therapeutic toxicity, and targeting of the diverse biomarkers present in the heterogeneous mixture of cells in primary and metastatic cancer. Theranostic use of radiolabeled SM binding many sites on many, not necessarily critical, biomarkers provides high cancer cell killing. Experiments to provide proof of principle of this novel concept suggested by the authors., (© 2018 WILEY Periodicals, Inc.)

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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA Encoded Libraries (DELs) use unique DNA sequences to tag each chemical warhead within a library mixture to enable deconvolution following affinity selection against a target protein. With next-generation sequencing, millions to billions of sequences can be read and counted to report binding events. This unprecedented capability has enabled researchers to synthesize and analyze numerically large chemical libraries. Despite the common perception that each library member undergoes a miniaturized affinity assay, selections with higher complexity libraries often produce results that are difficult to rank order. In this study, we aimed to understand the robustness of DEL selection by examining the sequencing readouts of warheads and chemotype families among a large number of experimentally repeated selections. The results revealed that (1) the output of DEL selection is intrinsically noisy but can be reliably modeled by the Poisson distribution, and (2) Poisson noise is the dominating noise at low copy counts and can be estimated even from a single experiment. We also discuss the shortcomings of data analyses based on directly using copy counts and their linear transformations, and propose a framework that incorporates proper normalization and confidence interval calculation to help researchers better understand DEL data.

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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have identified tris(2-aminoethyl)amine (tren)-derived scaffolds with two (t2M) or four (t4M) melamine rings that can target oligo T/U domains in DNA/RNA. Unstructured T-rich DNAs cooperatively fold with the tren derivatives to form hairpin-like structures. Both t2M and t4M act as functional switches in a family of hammerhead ribozymes deactivated by stem or loop replacement with a U-rich sequence. Catalysis of bond scission in these hammerhead ribozymes could be restored by putative t2M/t4M refolding of stem secondary structure or tertiary bridging interactions between loop and stem. The simplicity of the t2M/t4M binding site enables programming of allostery in RNAs, recoding oligo-U domains as potential sites for secondary structure or tertiary contact. In combination with a facile and general method for installation of the t2M motif on primary amines, the method described herein streamlines design of synthetic allosteric riboswitches and small molecule-nucleic acid complexes.

We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA-encoded chemical library (DEL) has emerged as a powerful and versatile tool for ligand discovery in chemical biology research and in drug discovery. Encoding and selection methods are two of the most important technological aspects of DEL that can dictate the performance and utilities of DELs. In this digest, we have summarized recent advances on the encoding and selection strategies of DEL and also discussed the latest developments on DNA-encoded dynamic library, a new frontier in DEL research., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs.

DNA display of PNA-encoded libraries was used to pair fragments containing different phosphotyrosine surrogates with diverse triazoles. Microarray-based screening of the combinatorially paired fragment sets (62,500 combinations) against a prototypical phosphatase, PTP1B, was used to identify the fittest fragments. A focused library (10,000 members) covalently pairing identified fragments with linkers of different length and geometry was synthesized. Screening of the focused library against PTP1B and closely related TCPTP revealed orthogonal inhibitors. The selectivity of the identified inhibitors for PTP1B versus TCPT was confirmed by enzymatic inhibition assay., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide-DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA-encoded chemical library (DECL) technology has emerged as a new avenue in the field of drug discovery. Combined with high-throughput sequencing, DECL selection experiments can provide not only many lead compounds but also insights into the structure-affinity relationship. However, the counts of individual DNA codes reflect, but cannot be used to precisely rank, the binding affinities of the corresponding compounds to protein targets. Herein, we describe a chip-based approach to realize an automated high-throughput assay for the kinetic characterization of the interaction between DNA-conjugated small organic compounds and protein targets. Importantly, this method can be applied to both single-pharmacophore DECLs and self-assembled dual-pharmacophore DECLs.

To efficiently identify small molecules binding to a G-quadruplex structure while avoiding binding to duplex DNA, we performed a multistep structure-based virtual screening by simultaneously taking into account G-quadruplex DNA and duplex DNA. Among the 13 compounds selected, one outstanding ligand shows significant selectivity for G-quadruplex binding as determined using SPR, FRET-based competition and luciferase activity assay.

Accumulating evidence implicating the role of aberrant transcription factor signaling in the pathogenesis of various human diseases such as cancer and inflammation has stimulated the development of small molecule ligands capable of targeting transcription factor activity and modulating gene expression. The use of DNA-binding small molecules to selectively inhibit transcription factor-DNA interactions represents one possible approach toward this goal. In this review, we summarize the development of DNA-binding small molecule inhibitors of transcription factors from 2004 to 2011, and their binding mode and therapeutic potential will be discussed., (© 2012 Wiley Periodicals, Inc.)

DNA methyltransferases (DNMTs) are emerging targets for the treatment of cancer and other diseases. The quinolone-based compound, SGI-1027, is a promising inhibitor of DNMT1 with a distinct mode of action and it is an attractive starting point for further research. Several experimental and computational approaches can be used to further develop novel DNMT1 inhibitors based on SGI-1027. In this work, we used a chemoinformatic-based approach to explore the potential to identify novel inhibitors in large screening collections of natural products and synthetic commercial libraries. Using the principles of similarity searching, the similarity profile to the active reference compound SGI-1027 was computed for four different screening libraries using a total of 22 two- and three- dimensional representations and two similarity metrics. The compound library with the overall highest similarity profile to the probe molecule was identified as the most promising collection for experimental testing. Individual compounds with high similarity to the reference were also selected as suitable candidates for experimental validation. During the course of this work, the 22 two- and three- dimensional representations were compared to each other and classified based on the similarity values computed with the reference compound. This classification is valuable to select structure representations for similarity searching of any other screening library. This work represents a step forward to further advance epigenetic therapies using computational approaches.

Three-way junction DNA (TWJ-DNA, also known as 3WJ-DNA) is an alternative secondary DNA structure comprised of three duplex-DNAs that converge towards a single point, termed the branch point. This point is characterized by unique geometrical properties that make its specific targeting by synthetic small-molecules possible. Such a targeting has already been demonstrated in the solid state but not thoroughly biophysically investigated in solution. Herein, a set of simple biophysical assays has been developed to identify TWJ-specific small-molecule ligands; these assays, inspired by the considerable body of work that has been reported to characterize the interactions between small-molecules and other higher-order DNA (notably quadruplex-DNA), have been calibrated with a known non-specific DNA binder (the porphyrin TMPyP4) and validated via the study of a small series of triazacyclononane (TACN) derivatives (metal-free or not) and the identification of a fairly-affinic and exquisitely TWJ-selective candidate (a TACN-quinoline construct named TACN-Q)., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Researchers seeking to improve the efficiency and cost effectiveness of the bioactive small-molecule discovery process have recently embraced selection-based approaches, which in principle offer much higher throughput and simpler infrastructure requirements compared with traditional small-molecule screening methods. Since selection methods benefit greatly from an information-encoding molecule that can be readily amplified and decoded, several academic and industrial groups have turned to DNA as the basis for library encoding and, in some cases, library synthesis. The resulting DNA-encoded synthetic small-molecule libraries, integrated with the high sensitivity of PCR and the recent development of ultra high-throughput DNA sequencing technology, can be evaluated very rapidly for binding or bond formation with a target of interest while consuming minimal quantities of material and requiring only modest investments of time and equipment. In this tutorial review we describe the development of two classes of approaches for encoding chemical structures and reactivity with DNA: DNA-recorded library synthesis, in which encoding and library synthesis take place separately, and DNA-directed library synthesis, in which DNA both encodes and templates library synthesis. We also describe in vitro selection methods used to evaluate DNA-encoded libraries and summarize successful applications of these approaches to the discovery of bioactive small molecules and novel chemical reactivity.