Expanding the DNA-encoded library toolbox: identifying small molecules targeting RNA

DNA-encoded library (DEL) technology is a powerful tool for small molecule identification in drug discovery, yet the reported DEL selection strategies were applied primarily on protein targets in either purified form or in cellular context. To expand the application of this technology, we employed DEL selection on an RNA target HIV-1 TAR (trans-acting responsive region), but found that the majority of signals were resulted from false positive DNA–RNA binding. We thus developed an optimized selection strategy utilizing RNA patches and competitive elution to minimize unwanted DNA binding, followed by k-mer analysis and motif search to differentiate false positive signal. This optimized strategy resulted in a very clean background in a DEL selection against Escherichia coli FMN Riboswitch, and the enriched compounds were determined with double digit nanomolar binding affinity, as well as similar potency in functional FMN competition assay. These results demonstrated the feasibility of small molecule identification against RNA targets using DEL selection. The developed experimental and computational strategy provided a promising opportunity for RNA ligand screening and expanded the application of DEL selection to a much wider context in drug discovery.     

DNA-encoded chemical libraries

The discovery and development of novel drugs for the multitude of targets originating from functional genomic research is a challenging task. While antibodies can nowadays be raised against virtually any given target using phage-display methodologies, a similar "selection/amplification" approach for the facile discovery of low-molecular weight compounds capable of specific binding to protein targets of choice has so far been lacking. The development of DNA-encoded chemical libraries, combined with suitable selection and high-throughput sequencing strategies, holds promises to fill this gap. Here, we review the latest developments in the field of DNA-encoded chemical libraries, commenting on the challenges and opportunities for the different experimental strategies in this rapidly evolving research area, which may gain importance for the future drug discovery process.     

Selection of streptavidin binders from a DNA-encoded chemical library

DNA-encoded libraries of small organic molecules facilitate the construction of large, encoded self-assembling chemical libraries for the identification of high-affinity binders to protein targets. We have constructed a library of 477 chemical compounds, coupled to 48mer-oligonucleotides, each containing a unique six-base sequence serving as "bar-code" for the identification of the chemical moiety. The functionality of the library was confirmed by selection and amplification of both high- and low-affinity binding molecules specific to streptavidin.     

High-throughput sequencing for the identification of binding molecules from DNA-encoded chemical libraries

DNA-encoded chemical libraries are large collections of small organic molecules, individually coupled to DNA fragments that serve as amplifiable identification bar codes. The isolation of specific binders requires a quantitative analysis of the distribution of DNA fragments in the library before and after capture on an immobilized target protein of interest. Here, we show how Illumina sequencing can be applied to the analysis of DNA-encoded chemical libraries, yielding over 10 million DNA sequence tags per flow-lane. The technology can be used in a multiplex format, allowing the encoding and subsequent sequencing of multiple selections in the same experiment. The sequence distributions in DNA-encoded chemical library selections were found to be similar to the ones obtained using 454 technology, thus reinforcing the concept that DNA sequencing is an appropriate avenue for the decoding of library selections. The large number of sequences obtained with the Illumina method now enables the study of very large DNA-encoded chemical libraries (>500,000 compounds) and reduces decoding costs.     

Design and synthesis of a novel DNA-encoded chemical library using Diels-Alder cycloadditions

DNA-encoded chemical libraries are increasingly being employed for the identification of binding molecules to protein targets of pharmaceutical relevance. Here, we describe the synthesis and characterization of a DNA-encoded chemical library, consisting of 4000 compounds generated by Diels-Alder cycloaddition reactions. The compounds were encoded with unique DNA fragments which were generated through a stepwise assembly process and serve as amplifiable bar codes for the identification and relative quantification of library members.     

Application of encoded library technology (ELT) to a protein–protein interaction target: Discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists

The inhibition of protein–protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein.     

Integrating DNA-encoded chemical libraries with virtual combinatorial library screening: Optimizing a PARP10 inhibitor

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.     

Advances in Lead Generation

Lead Generation represents a critical drug discovery phase where chemical starting points and their respective mechanism of action, quality, and potential liabilities are largely predefined. Recent advances such as DNA-encoded libraries or fragment-, chemical biology-, and virtual screening-based approaches are today as common as traditional High Throughput Screening. Innovations in characterizing lead quality have allowed more informed decision-making by discovery teams. The key challenge today is to individually tailor the right mix of methods for each project to facilitate data integration with the purpose of creating multiple high-quality lead series, ultimately translating to reduced chemistry-related pipeline attrition.     

生物催化剂发现与改造的研究进展

生物催化是指将酶或生物有机体用于有用的化学转化的过程,在人们对传统化学催化的环境影响抱有忧虑的情况下,生物催化提供了一种有吸引力的选择。在过去的几十年里,对生物催化剂的研究每出现一次大的进步,生物催化的发展就会出现一次高潮。因此,生物催化剂的发现与改造已成为当今研究的热点。宏基因组文库技术的出现克服了许多微生物不可培养的障碍,人们能够从自然资源中获得丰富的潜在的生物催化剂。而基于理性设计的分子改造技术的发展,可以使得人们对潜在的生物催化剂进行快速而有效的改造以满足工业化生产的需求。随着生物催化剂发现与改造的手段不断进步,更多的优良生物催化剂得到了广泛的应用,生物催化在工业生产中也得到了更深入的应用。结合作者的研究工作,总结了生物催化剂发现与改良的一些研究进展,以为获得更多优良的、能够实现工业应用的生物催化剂奠定理论基础。     

Navigating large chemical spaces in early-phase drug discovery

The size of actionable chemical spaces is surging, owing to a variety of novel techniques, both computational and experimental. As a consequence, novel molecular matter is now at our fingertips that cannot and should not be neglected in early-phase drug discovery. Huge, combinatorial, make-on-demand chemical spaces with high probability of synthetic success rise exponentially in content, generative machine learning models go hand in hand with synthesis prediction, and DNA-encoded libraries offer new ways of hit structure discovery. These technologies enable to search for new chemical matter in a much broader and deeper manner with less effort and fewer financial resources.These transformational developments require new cheminformatics approaches to make huge chemical spaces searchable and analyzable with low resources, and with as little energy consumption as possible. Substantial progress has been made in the past years with respect to computation as well as organic synthesis. First examples of bioactive compounds resulting from the successful use of these novel technologies demonstrate their power to contribute to tomorrow's drug discovery programs. This article gives a compact overview of the state-of-the-art.     

噬菌体抗体库技术在肿瘤抗体药物研究中的进展及应用

目的:综述噬菌体抗体库技术的研究进展,介绍该技术的原理,构建,筛选和应用,为抗肿瘤抗体药物研发提供参考。方法:采用文献综述的方法,筛选近5年来噬菌体抗体库技术试验论文,对噬菌体抗体库技术的原理,构建,筛选和应用进行总结。结果:噬菌体抗体库主要分为免疫抗体库和非免疫抗体库两大类;噬菌体抗体库筛选技术包括亲和筛选、细胞筛选和生物体内筛选三种;噬菌体抗体库技术主要应用于肿瘤标志物的识别和肿瘤诊断,抗肿瘤抗体药物的筛选和制备。结论:噬菌体抗体库技术方便、快速、高效,可以在体外环境下培养,这些特点决定了其在肿瘤标志物的发现和肿瘤抗体药物研发中的广泛应用。目前噬菌体抗体库技术还存在一定缺陷,但技术的不断发展和革新必然使噬菌体抗体库技术成为研制抗体药物的新思路,极大促进了肿瘤抗体药物的研发。     

DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA "genes," making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This "chemical translation" provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science "biology."     

Yeast DEL assay detects protection against radiation-induced cytotoxicity and genotoxicity: adaptation of a microtiter plate version

The DEL assay in yeast detects DNA deletions that are inducible by many carcinogens. Here we use the colorimetric agent MTS to adapt the yeast DEL assay for microwell plate measurement of ionizing radiation-induced cell killing and DNA deletions. Using the microwell-based DEL assay, cell killing and genotoxic DNA deletions both increased with radiation dose between 0 and 2000 Gy. We used the microwell-based DEL assay to assess the effectiveness of varying concentrations of five different radioprotectors, N-acetyl-l-cysteine, l-ascorbic acid, DMSO, Tempol and Amifostine, and one radiosensitizer, 5-bromo-2-deoxyuridine. The microwell format of the DEL assay was able to successfully detect protection against and sensitization to both radiation-induced cytotoxicity and genotoxicity. Such radioprotection and sensitization detected by the microwell-based DEL assay was validated and compared with similar measurements made using the traditional agar-based assay format. The yeast DEL assay in microwell format is an effective tool for rapidly detecting chemical protectors and sensitizers to ionizing radiation and is automatable for chemical high-throughput screening purposes.     

Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity

Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for therapeutic and other applications. Antibody clones from synthetic libraries with optimized design features have several distinct advantages that include high stability, high levels of expression, and ease of downstream optimization and engineering. In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides. In order to maximize the functional diversity of the library, a β-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5′-end of the β-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of 7.6 × 10⁹, greater than 70% functional diversity, and diversification of all six CDRs was obtained after insertion of fully randomized CDR-H3 sequences into this proofread repertoire. The performance of the library was validated using a number of target antigens, against which multiple unique scFv sequences with dissociation constants in the nanomolar range were isolated.     

哺乳动物细胞小干扰RNA库的建立和应用

双链小干扰RNA（siRNA）在多种类型细胞中介导特异性的基因沉默,这一现象的发现为深入研究单个基因的功能提供了重要的方法学基础,从而得到了广泛的应用.最近的文献报道了全基因组siRNA库的建立,为高通量基因功能分析和研究提供了新的方法,成为新的研究热点.小干扰RNA库可以用来筛选和研究介导细胞复杂表型和生物学过程的关键基因,通过建立一系列具有目的表型的细胞系,有可能对特定细胞信号调节通路进行更为全面的解析.本文综述了目前在siRNA建库方法方面的进展,并探讨了建立小干扰RNA库中的关键问题.     

Role of natural product diversity in chemical biology

Through the natural selection process, natural products possess a unique and vast chemical diversity and have been evolved for optimal interactions with biological macromolecules. Owing to their diversity, target affinity, and specificity, natural products have demonstrated enormous potential as modulators of biomolecular function, been an essential source for drug discovery, and provided design principles for combinatorial library development.     

Discovery of inhibitors of the pentein superfamily protein dimethylarginine dimethylaminohydrolase (DDAH), by virtual screening and hit analysis

An efficient process for the discovery of inhibitors of DDAH enzymes, without the requirement for high throughput screening, is described. Physicochemical filtering of a 308,000-compound library according to drug likeness followed by reciprocal nearest neighbour selection produced a representative subset of 35,000 compounds. Virtual screening on a dual processor PC using FlexX, followed by biological screening, identified two hit series. Similarity searches of commercial databases and chemical re-synthesis of pure compounds resulted in SR445 as an inhibitor of Pseudomonas aeruginosa DDAH at 2 microM.     

A surface display yeast two-hybrid screening system for high-throughput protein interactome mapping

Despite the wide acceptance of yeast two-hybrid (Y2H) system for protein-protein interaction analysis and discovery, conventional Y2H assays are not well suited for high-throughput screening of the protein interaction network (“interactome”) on a genomic scale due to several limitations, including labor-intensive agar plating and colony selection methods associated with the use of nutrient selection markers, complicated reporter analysis methods associated with the use of LacZ enzyme reporters, and incompatibility of the liquid handling robots. We recently reported a robust liquid culture Y2H system based on quantitative analysis of yeast-enhanced green fluorescent protein (yEGFP) reporters that greatly increased the analysis throughput and compatibility with liquid handling robots. To further advance its utility in high-throughput complementary DNA (cDNA) library screening, we report the development of a novel surface display Y2H (sdY2H) library screening system with uniquely integrated surface display hemagglutination (sdHA) antigen and yEGFP reporters. By introduction of a surface reporter sdHA into the yEGFP-based Y2H system, positive Y2H targets are quickly isolated from library cells by a simple magnetic separation without a large plating effort. Moreover, the simultaneous scoring of multiple reporters, including sdHA, yEGFP, and conventional nutrient markers, greatly increased the specificity of the Y2H assay. The feasibility of the sdY2H assay on large cDNA library screening was demonstrated by the successful recovery of positive P53/T interaction pairs at a target-to-background ratio of 1:1,000,000. Together with the massive parallel DNA sequencing technology, it may provide a powerful proteomic tool for high-throughput interactome mapping on a genomic scale.