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.     

Forward chemical genetics: library scaffold design

With the unraveling of the entire human genome, it has become imperative to understand the function of the gene products, proteins. Within the past several years, chemical genetics has gained recognition as a powerful approach to study protein function by using small molecules as gene knock-out or knock-in mimics. Forward chemical genetics is a three-step process; the design and synthesis of a small molecule library represents the first step followed secondly by the search for novel phenotypes and then by isolation and identification of target protein(s). This review will focus on the first step, the design of the scaffold for small molecule libraries. It will also examine the connection between the choice of a scaffold and the propensity of that library to demonstrate enhanced biological activity when tested in certain cellular systems.     

SAR by ILOEs: an NMR-based approach to reverse chemical genetics

Reverse chemical genetics is an emerging technique that makes use of small molecule inhibitors to characterize how a protein functions. In this regard, we have developed an NMR-based approach (SAR by ILOEs) that enables the identification of high affinity ligands for a given protein target without the need of a specific assay. Our approach is of general applicability and could result very powerful in reverse chemical-genetics studies, target validation, and lead discovery. We report a recent application on the design and synthesis of compounds that inhibit protein-membrane interactions.     

Quality, not quantity: the role of natural products and chemical proteomics in modern drug discovery

Chemical genetics and reverse chemical genetics parallel classical genetics but target genes at the protein level and have proven useful in recent years for screening combinatorial libraries for compounds of biological interest. However, the performance of combinatorial chemistry in filling pharmaceutical pipelines has been lower than anticipated and the tide may be turning back to Nature in the search for new drug candidates. Even though diversity oriented synthesis is now producing molecules that are natural product-like in terms of size and complexity, these molecules have not evolved to interact with biomolecules. Natural products, on the other hand, have evolved to interact with biomolecules, which is why so many can be found in pharmacopoeias. However, the cellular targets and modes of action of these fascinating compounds are seldom known, hindering the drug development process. This review focuses on the emergence of chemical proteomics and reverse chemical proteomics as tools for the discovery of cellular receptors for natural products, thereby generating protein/ligand pairs that will prove useful in identifying new drug targets and new biologically active small molecule scaffolds. Such a system-wide approach to identifying new drugable targets and their small molecule ligands will help unblock the pharmaceutical product pipelines by speeding the process of target and lead identification.     

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment–protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.     

Image‐Based Morphological Profiling Identifies a Lysosomotropic,Iron‐Sequestering Autophagy Inhibitor

Chemical proteomics is widely applied in small‐molecule target identification. However, in general it does not identify non‐protein small‐molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image‐based morphological profiling in the cell painting assay. A compound‐induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe²⁺ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.     

Ligand design by a combinatorial approach based on modeling and experiment: application to HLA-DR4

Combinatorial synthesis and large scale screening methods are being used increasingly in drug discovery, particularly for finding novel lead compounds. Although these "random" methods sample larger areas of chemical space than traditional synthetic approaches, only a relatively small percentage of all possible compounds are practically accessible. It is therefore helpful to select regions of chemical space that have greater likelihood of yielding useful leads. When three-dimensional structural data are available for the target molecule this can be achieved by applying structure-based computational design methods to focus the combinatorial library. This is advantageous over the standard usage of computational methods to design a small number of specific novel ligands, because here computation is employed as part of the combinatorial design process and so is required only to determine a propensity for binding of certain chemical moieties in regions of the target molecule. This paper describes the application of the Multiple Copy Simultaneous Search (MCSS) method, an active site mapping and de novo structure-based design tool, to design a focused combinatorial library for the class II MHC protein HLA-DR4. Methods for the synthesizing and screening the computationally designed library are presented; evidence is provided to show that binding was achieved. Although the structure of the protein-ligand complex could not be determined, experimental results including cross-exclusion of a known HLA-DR4 peptide ligand (HA) by a compound from the library. Computational model building suggest that at least one of the ligands designed and identified by the methods described binds in a mode similar to that of native peptides.     

化学遗传学简介

化学遗传学是20世纪90年代开始兴起的交叉学科,是利用生物活性小分子与蛋白相互作用研究生物学系统功能的一种方法,是经典遗传学的补充。化学遗传学的历史可以追溯到几百年前。在现代药物靶标的发现上,化学遗传学起着非常重要的作用。     

Enhancement of chemical rules for predicting compound reactivity towards protein thiol groups

Non-specific chemical modification of protein thiol groups continues to be a significant source of false positive hits from high-throughput screening campaigns and can even plague certain protein targets and chemical series well into lead optimization. While experimental tools exist to assess the risk and promiscuity associated with the chemical reactivity of existing compounds, computational tools are desired that can reliably identify substructures that are associated with chemical reactivity to aid in triage of HTS hit lists, external compound purchases, and library design. Here we describe a Bayesian classification model derived from more than 8,800 compounds that have been experimentally assessed for their potential to covalently modify protein targets. The resulting model can be implemented in the large-scale assessment of compound libraries for purchase or design. In addition, the individual substructures identified as highly reactive in the model can be used as look-up tables to guide chemists during hit-to-lead and lead optimization campaigns.     

Photoreactive stapled BH3 peptides to dissect the BCL-2 family interactome

Defining protein interactions forms the basis for discovery of biological pathways, disease mechanisms, and opportunities for therapeutic intervention. To harness the robust binding affinity and selectivity of structured peptides for interactome discovery, we engineered photoreactive stapled BH3 peptide helices that covalently capture their physiologic BCL-2 family targets. The crosslinking α helices covalently trap both static and dynamic protein interactors, and enable rapid identification of interaction sites, providing a critical link between interactome discovery and targeted drug design.     

Image-Based Morphological Profiling Identifies a Lysosomotropic,Iron-Sequestering Autophagy Inhibitor

Chemical proteomics is widely applied in small-molecule target identification. However, in general it does not identify non-protein small-molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image-based morphological profiling in the cell painting assay. A compound-induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe²⁺ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.     

Target identification strategies in chemical genetics

Chemical inhibitors have had a profound impact on many diverse fields of biology. The goal of chemical genetics is to use small molecules to perturb biological systems in a manner conceptually similar to traditional genetics. Key to the advancement of the chemical genetic paradigm is the further development of tools and approaches for the identification of the protein targets of active compounds identified in chemical genetic screens. This review will address historic examples in which forward chemical genetics yielded new insight into a biological problem through successful identification of the target of an active molecule. The approaches covered have been grouped into two broad classes: target identification by affinity-based methods and target identification by deduction. Strengths and shortcomings of each approach as it pertains to their application to modern chemical genetics will be discussed. Finally, a series of new genomic and proteomic-based techniques for target identification will be described. Although a truly general approach to target identification has yet to be developed, these examples illustrate that there are many effective strategies for successfully elucidating the biological targets of active small molecules.     

活性导向的化学探针在氨基酸反应活性表征中的应用进展

原创药物的研制得益于蛋白质新靶标的发现,而新靶标的发现依赖于高可信度、高通量的药物-蛋白质相互作用分析方法。蛋白质作为生命功能的执行者,其表达量、空间定位与结构差异直接影响药效的发挥。目前,超过85%的蛋白质尚被认为是无法成药的,主要原因是缺少药物分子靶向的空腔以及相应的反应活性位点。因此,基于蛋白质组学层次实现对氨基酸反应活性位点的表征成为原创共价靶向药物设计的关键,也是克服难以成药靶标蛋白问题的关键。近年来,质谱技术的飞速发展极大地推动了基于蛋白质组学技术的药物-靶蛋白相互作用研究。其中基于活性的蛋白质组分析(ABPP)策略是利用活性位点导向的化学探针分子在复杂样品中实现功能状态酶和药物靶标等蛋白质的检测。基于化学探针的开发和质谱定量技术的发展,ABPP技术在氨基酸反应活性表征研究中展现出重要的应用潜力,将助力于药物新靶标的发现和药物先导化合物的开发。ABPP策略主要基于蛋白质的活性特征进行富集,活性探针作为ABPP策略的核心,近年来取得了飞速进展。该文回顾了ABPP策略的发展历程,重点介绍基于广谱活性探针的ABPP技术在多种氨基酸反应活性筛选领域的研究进展,并对其在药物靶点发现中...     

Synthesis and screening of peptoid arrays on cellulose membranes

Rapid synthesis and screening of compound libraries enables the accelerated identification of novel protein ligands in order to support processes like analysis of protein interactions, drug target discovery or lead structure discovery. SPOT synthesis—a well established method for the rapid preparation of peptide arrays—has recently been extended to the field of nonpeptides. In this contribution we report on the systematic evaluation of the SPOT technique for the assembly of N-alkylglycine (peptoid) library arrays. In the course of this investigation bromoacetic acid 2,4-dinitrophenylester (1a) was identified to be the most suited agent for bromoacetylation in terms of yield and N-selectivity enabling straightforward submonomer synthesis on hydroxy-group rich cellulose membranes. The potential of this method for the rapid identification of novel nonpeptidic protein ligands was demonstrated by synthesis and screening of a library consisting of 8000 peptoids and peptomers (i.e. their hybrids with α-substituted amino acids) allowing the identification of micromolar ligands for the monoclonal antibody Tab-2.     

Modular synthesis and preliminary biological evaluation of stereochemically diverse 1,3-dioxanes

Modular synthesis and substrate stereocontrol were combined to furnish 18,000 diverse 1,3-dioxanes whose distribution in chemical space rivals that of a reference set of over 2,000 bioactive small molecules. Library quality was assessed at key synthetic stages, culminating in a detailed postsynthesis analysis of purity, yield, and structural characterizability, and the resynthesis of library subsets that did not meet quality standards. The importance of this analysis-resynthesis process is highlighted by the discovery of new biological probes through organismal and protein binding assays, and by determination of the building block and stereochemical basis for their bioactivity. This evaluation of a portion of the 1,3-dioxane library suggests that many additional probes for chemical genetics will be identified as the entire library becomes biologically annotated.     

From Phenotypic Hit to Chemical Probe: Chemical Biology Approaches to Elucidate Small Molecule Action in Complex Biological Systems

Biologically active small molecules have a central role in drug development, and as chemical probes and tool compounds to perturb and elucidate biological processes. Small molecules can be rationally designed for a given target, or a library of molecules can be screened against a target or phenotype of interest. Especially in the case of phenotypic screening approaches, a major challenge is to translate the compound-induced phenotype into a well-defined cellular target and mode of action of the hit compound. There is no “one size fits all” approach, and recent years have seen an increase in available target deconvolution strategies, rooted in organic chemistry, proteomics, and genetics. This review provides an overview of advances in target identification and mechanism of action studies, describes the strengths and weaknesses of the different approaches, and illustrates the need for chemical biologists to integrate and expand the existing tools to increase the probability of evolving screen hits to robust chemical probes.     

Diversified strategy for the synthesis of DNA-encoded oxindole libraries

DNA-encoded library technology (DELT) employs DNA as a barcode to track the sequence of chemical reactions and enables the design and synthesis of libraries with billions of small molecules through combinatorial expansion. This powerful technology platform has been successfully demonstrated for hit identification and target validation for many types of diseases. As a highly integrated technology platform, DEL is capable of accelerating the translation of synthetic chemistry by using on-DNA compatible reactions or off-DNA scaffold synthesis. Herein, we report the development of a series of novel on-DNA transformations based on oxindole scaffolds for the design and synthesis of diversity-oriented DNA-encoded libraries for screening. Specifically, we have developed 1,3-dipolar cyclizations, cyclopropanations, ring-opening of reactions of aziridines and Claisen–Schmidt condensations to construct diverse oxindole derivatives. The majority of these transformations enable a diversity-oriented synthesis of DNA-encoded oxindole libraries which have been used in the successful hit identification for three protein targets. We have demonstrated that a diversified strategy for DEL synthesis could accelerate the application of synthetic chemistry for drug discovery.

Constructing DNA-encoded oxindole libraries by a diversified strategy.     

High‐Throughput Kinetic Analysis for Target‐Directed Covalent Ligand Discovery

Cysteine‐reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high‐quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan‐reactive compounds. Quantitative irreversible tethering (qIT), a general method for screening cysteine‐reactive small molecules based upon the maximization of kinetic selectivity, is described. This method was applied prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2‐selective allosteric (type IV) kinase inhibitor whose novel mode‐of‐action could be exploited therapeutically.     

基于化学蛋白质组学的药物靶标鉴定

通过药物靶标鉴定，可以建立药物活性与细胞表型之间的联系，阐明药物的作用机理，还可以发现药物的脱靶效应和耐药性机制，发现治疗药物的新靶点，并在药物开发的早期阶段预测可能存在的毒性，降低药物研发失败的风险。目前虽然科学技术取得了飞速发展，但是鉴定药物靶标依然是一件令人生畏的工作。本文对近10年来药物靶标鉴定方面的研究进展，特别是无化学标记的新技术进行了评述。     

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.