DNA-Encoded Macrocyclic Peptide Library.

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.

Development of a Selection Method for Discovering Irreversible (Covalent) Binders from a DNA-Encoded Library.

DNA-encoded libraries (DELs) have been broadly applied to identify chemical probes for target validation and lead discovery. To date, the main application of the DEL platform has been the identification of reversible ligands using multiple rounds of affinity selection. Irreversible (covalent) inhibition offers a unique mechanism of action for drug discovery research. In this study, we report a developing method of identifying irreversible (covalent) ligands from DELs. The new method was validated by using 3C protease (3CP) and on-DNA irreversible tool compounds (rupintrivir derivatives) spiked into a library at the same concentration as individual members of that library. After affinity selections against 3CP, the irreversible tool compounds were specifically enriched compared with the library members. In addition, we compared two immobilization methods and concluded that microscale columns packed with the appropriate affinity resin gave higher tool compound recovery than magnetic beads.

Author Correction: Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening.

This corrects the article DOI: 10.1038/ncomms16081.

Design and Application of a DNA-Encoded Macrocyclic Peptide Library.

A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening.

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

Ruthenium Promoted On-DNA Ring-Closing Metathesis and Cross-Metathesis.

DNA-encoded library technology (ELT) is now widely used in pharmaceutical, biotechnological, and academic research for hit identification and target validation. New on-DNA reactions are keys to exploring greater chemical space and accessing challenging chemotypes such as configurationally constrained macrocycles. Herein, we describe the first on-DNA ring-closing metathesis (RCM) and cross-metathesis (CM) reactions promoted by fast initiating Grubbs Ru reagents. Under the optimized conditions, MgCl2 was used to protect the DNA from Ru-induced decomposition. The substrate scope for on-DNA RCM was established and the same conditions were applied to a CM reaction with good conversion.

Application of Biocatalysis to on-DNA Carbohydrate Library Synthesis.

DNA-encoded libraries are increasingly used for the discovery of bioactive lead compounds in high-throughput screening programs against specific biological targets. Although a number of libraries are now available, they cover limited chemical space due to bias in ease of synthesis and the lack of chemical reactions that are compatible with DNA tagging. For example, compound libraries rarely contain complex biomolecules such as carbohydrates with high levels of functionality, stereochemistry, and hydrophilicity. By using biocatalysis in combination with chemical methods, we aimed to significantly expand chemical space and generate generic libraries with potentially better biocompatibility. For DNA-encoded libraries, biocatalysis is particularly advantageous, as it is highly selective and can be performed in aqueous environments, which is an essential feature for this split-and-mix library technology. In this work, we demonstrated the application of biocatalysis for the on-DNA synthesis of carbohydrate-based libraries by using enzymatic oxidation and glycosylation in combination with traditional organic chemistry.

On-DNA Pd and Cu promoted C-N cross-coupling reactions.

Encoded library technology (ELT) is a novel hit identification platform synergistic with HTS, fragment hit ID and focused screening. It provides both an ultra high-throughput and a cost-efficient tool for the discovery of small molecules that bind to protein targets of pharmaceutical interest. The success of ELT relies heavily on the chemical diversity accessed through DNA-encoded library (DEL) synthesis. We developed unprecedented Pd and Cu(i) promoted C-N cross-coupling reactions between a DNA-conjugated aryl iodide and primary amines. These reported reactions have strong potential for application in DNA-encoded library (DEL) synthesis.

Zirconium(IV)-Catalyzed Ring Opening of on-DNA Epoxides in Water.

DNA-encoded library technology (ELT) has spurred wide interest in the pharmaceutical industry as a powerful tool for hit and lead generation. In recent years a number of "DNA-compatible" chemical modifications have been published and used to synthesize vastly diverse screening libraries. Herein we report a newly developed, zirconium tetrakis(dodecyl sulfate) [Zr(DS)4 ] catalyzed ring-opening of on-DNA epoxides in water with amines, including anilines. Subsequent cyclization of the resulting on-DNA ß-amino alcohols leads to a variety of biologically interesting, nonaromatic heterocycles. Under these conditions, a library of 137 million on-DNA ß-amino alcohols and their cyclization products was assembled.

Discovery, SAR, and X-ray Binding Mode Study of BCATm Inhibitors from a Novel DNA-Encoded Library.

As a potential target for obesity, human BCATm was screened against more than 14 billion DNA encoded compounds of distinct scaffolds followed by off-DNA synthesis and activity confirmation. As a consequence, several series of BCATm inhibitors were discovered. One representative compound (R)-3-((1-(5-bromothiophene-2-carbonyl)pyrrolidin-3-yl)oxy)-N-methyl-2'-(methylsulfonamido)-[1,1'-biphenyl]-4-carboxamide (15e) from a novel compound library synthesized via on-DNA Suzuki-Miyaura cross-coupling showed BCATm inhibitory activity with IC50 = 2.0 µM. A protein crystal structure of 15e revealed that it binds to BCATm within the catalytic site adjacent to the PLP cofactor. The identification of this novel inhibitor series plus the establishment of a BCATm protein structure provided a good starting point for future structure-based discovery of BCATm inhibitors.

Encoded library technology as a source of hits for the discovery and lead optimization of a potent and selective class of bactericidal direct inhibitors of Mycobacterium tuberculosis InhA.

Tuberculosis (TB) is one of the world's oldest and deadliest diseases, killing a person every 20 s. InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, is the target of the frontline antitubercular drug isoniazid (INH). Compounds that directly target InhA and do not require activation by mycobacterial catalase peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. The application of the encoded library technology (ELT) to the discovery of direct InhA inhibitors yielded compound 7 endowed with good enzymatic potency but with low antitubercular potency. This work reports the hit identification, the selected strategy for potency optimization, the structure-activity relationships of a hundred analogues synthesized, and the results of the in vivo efficacy studies performed with the lead compound 65.

Discovery of highly potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded library technology (ELT).

The metalloprotease ADAMTS-5 is considered a potential target for the treatment of osteoarthritis. To identify selective inhibitors of ADAMTS-5, we employed encoded library technology (ELT), which enables affinity selection of small molecule binders from complex mixtures by DNA tagging. Selection of ADAMTS-5 against a four-billion member ELT library led to a novel inhibitor scaffold not containing a classical zinc-binding functionality. One exemplar, (R)-N-((1-(4-(but-3-en-1-ylamino)-6-(((2-(thiophen-2-yl)thiazol-4-yl)methyl)amino)-1,3,5-triazin-2-yl)pyrrolidin-2-yl)methyl)-4-propylbenzenesulfonamide (8), inhibited ADAMTS-5 with IC(50) = 30 nM, showing >50-fold selectivity against ADAMTS-4 and >1000-fold selectivity against ADAMTS-1, ADAMTS-13, MMP-13, and TACE. Extensive SAR studies showed that potency and physicochemical properties of the scaffold could be further improved. Furthermore, in a human osteoarthritis cartilage explant study, compounds 8 and 15f inhibited aggrecanase-mediated (374)ARGS neoepitope release from aggrecan and glycosaminoglycan in response to IL-1ß/OSM stimulation. This study provides the first small molecule evidence for the critical role of ADAMTS-5 in human cartilage degradation.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

Synthesis of 2-indanones via [4 + 1] annulation reactions of (trialkylsilyl)arylketenes.

[reaction: see text] (Trialkylsilyl)arylketenes combine with (trimethylsilyl)diazomethane in a new [4 + 1] annulation process leading to 2-indanone derivatives. The (trialkylsilyl)arylketene annulation substrates are available via the photochemical Wolff rearrangement of alpha-silyl-alpha-diazo ketones, which are themselves prepared by silylation of the corresponding diazo ketones. The mechanism of the annulation reaction is proposed to involve the formation of a 2,3-bis(silyl)cyclopropanone, which is in equilibrium with an oxyallylic cation. Electrocyclic closure of this intermediate forms the new cyclopentenone ring.