Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library

Litovchick, Alexander; Dumelin, Christoph E.; Habeshian, Sevan; Gikunju, Diana; Guié, Marie-Aude; Centrella, Paolo A.; Zhang, Ying; Sigel, Eric A.; Cuozzo, John W.; Keefe, Anthony D.; Clark, Matthew

doi:10.1038/srep10916

Cited by 103 publications

(121 citation statements)

References 30 publications

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“…(8,(16)(17)(18). InhA containing an N-terminal 6xHis tag was mixed with the library pool in the presence or absence of NADH or NAD + .…”

Section: Significancementioning

confidence: 99%

Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology

Soutter¹,

Centrella²,

Clark

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl–acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (1011 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

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“…(8,(16)(17)(18). InhA containing an N-terminal 6xHis tag was mixed with the library pool in the presence or absence of NADH or NAD + .…”

Section: Significancementioning

confidence: 99%

Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology

Soutter¹,

Centrella²,

Clark

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…A potent inhibitor of soluble epoxide hydrolase (sEH) was also developed by X-Chem using an innovative DECL encoding strategy, featuring a DNA-ligation step that made use of Cu-catalyzed alkyne-azide cycloaddition (CuAAC) [59]. A 334-million member library was synthesized using three sets of building blocks (2259 primary amines, 222 bromoaryl acids, and 667 boronic acids).…”

Section: Ligands Isolated From Dna-encoded Chemical Librariesmentioning

confidence: 99%

DNA‐encoded chemical libraries – achievements and remaining challenges

et al. 2018

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Edited by Wilhelm JustDNA-encoded chemical libraries (DECLs) are collections of compounds, individually coupled to DNA tags serving as amplifiable identification barcodes. Since individual compounds can be identified by the associated DNA tag, they can be stored as a mixture, allowing the synthesis and screening of combinatorial libraries of unprecedented size, facilitated by the implementation of split-and-pool synthetic procedures or other experimental methodologies. In this review, we briefly present relevant concepts and technologies, which are required for the implementation and interpretation of screening procedures with DNA-encoded chemical libraries. Moreover, we illustrate some success stories, detailing how novel ligands were discovered from encoded libraries. Finally, we critically review what can realistically be achieved with the technology at the present time, highlighting challenges and opportunities for the future.

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“…For standard RNAseq experiments such as the one described here, this is not an issue as we already under-sample the original RNA by multiple orders of magnitude (100 ng FHV RNA would correspond to 2 Trillion 100 nt reads at 1 X coverage). For applications requiring a thorough capture of the original RNA such as single-cell or single-molecule studies 40; 41 , the efficiency of the click-reaction and the subsequent read-though must be improved, for example, by using evolved or rationally-designed polymerases in optimized buffer conditions 42 .…”

mentioning

confidence: 99%

ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3′-Azido cDNAs

Routh

Head

Ordoukhanian

et al. 2015

Journal of Molecular Biology

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We present a simple method called ‘ClickSeq’ for Next-Generation Sequencing (NGS) library synthesis that uses click-chemistry rather than enzymatic reactions for the ligation of Illumina sequencing adaptors. In ClickSeq, randomly-primed reverse transcription reactions are supplemented with azido-2’,3’-dideoxynucleotides that randomly terminate DNA synthesis and release 3’-azido blocked cDNA fragments in a process akin to dideoxy-sanger sequencing. Purified fragments are ‘click-ligated’ via copper-catalyzed alkyne-azide cycloaddition to DNA oligos modified with a 5’-alkyne group. This generates ssDNA molecules containing an unnatural triazole-linked DNA backbone that is sufficiently biocompatible for PCR amplification to generate a cDNA library for RNAseq. Here, we analyze viral RNAs and mRNA to demonstrate that ClickSeq produces unbiased NGS libraries with low error-rates comparable to standard methods. Importantly, ClickSeq is robust against common artifacts of NGS such as chimera formation and artifactual recombination with fewer than 3 aberrant events detected per million reads.

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Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library

Cited by 103 publications

References 30 publications

Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology

Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology

DNA‐encoded chemical libraries – achievements and remaining challenges

ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3′-Azido cDNAs

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