Tools and methodologies capable of isolating and identifying a target molecule for a bioactive compound

Elucidating the mechanism of action of bioactive compounds, such as commonly used pharmaceutical drugs and biologically active natural products, in the cells and the living body is important in drug discovery research. To this end, isolation and identification of target protein(s) for the bioactive compound are essential in understanding its function fully. And, development of reliable tools and methodologies capable of addressing efficiently identification and characterization of the target proteins based on the bioactive compounds accelerates drug discovery research. Affinity-based isolation and identification of target molecules for the bioactive compounds is a classic, but still powerful approach. This paper introduces recent progress on affinity chromatography system, focusing on development of practical affinity matrices and useful affinity-based methodologies on target identification. Beneficial affinity chromatography systems with using practical tools and useful methodologies facilitate chemical biology and drug discovery research.     

Identification and validation of bioactive small molecule target through phenotypic screening

For effective bioactive small molecule discovery and development into new therapeutic drug, a systematic screening and target protein identification is required. Different from the conventional screening system, herein phenotypic screening in combination with multi-omics-based target identification and validation (MOTIV) is introduced. First, phenotypic screening provides visual effect of bioactive small molecules in the cell or organism level. It is important to know the effect on the cell or organism level since small molecules affect not only a single target but the entire cellular mechanism within a cell or organism. Secondly, MOTIV provides systemic approach to discover the target protein of bioactive small molecule. With the chemical genomics and proteomics approach of target identification methods, various target protein candidates are identified. Then network analysis and validations of these candidates result in identifying the biologically relevant target protein and cellular mechanism. Overall, the combination of phenotypic screening and MOTIV will provide an effective approach to discover new bioactive small molecules and their target protein and mechanism identification.     

Proteomic methods for drug target discovery

The field of drug target discovery is currently very popular with a great potential for advancing biomedical research and chemical genomics. Innovative strategies have been developed to aid the process of target identification, either by elucidating the primary mechanism-of-action of a drug, by understanding side effects involving unanticipated 'off-target' interactions, or by finding new potential therapeutic value for an established drug. Several promising proteomic methods have been introduced for directly isolating and identifying the protein targets of interest that are bound by active small molecules or for visualizing enzyme activities affected by drug treatment. Significant progress has been made in this rapidly advancing field, speeding the clinical validation of drug candidates and the discovery of the novel targets for lead compounds developed using cell-based phenotypic screens. Using these proteomic methods, further insight into drug activity and toxicity can be ascertained.     

A protein domain interaction interface database: InterPare

Background  

Most proteins function by interacting with other molecules. Their interaction interfaces are highly conserved throughout evolution to avoid undesirable interactions that lead to fatal disorders in cells. Rational drug discovery includes computational methods to identify the interaction sites of lead compounds to the target molecules. Identifying and classifying protein interaction interfaces on a large scale can help researchers discover drug targets more efficiently.     

Target deconvolution from phenotype-based drug discovery by using chemical proteomics approaches

Phenotype-based drug discovery is a key strategy for small molecule drug screening, and the molecular target identification of small molecules, termed “target deconvolution,” is critical albeit challenging. In this review, we classify approaches for target deconvolution, including both direct and indirect approaches, summarize their underlying principles, and provide examples of current chemical proteomics strategies including affinity purification using compound-immobilized beads, photoaffinity labeling (PAL), cellular thermal shift assay (CETSA), and activity-based protein profiling (ABPP). Because there is no single best target deconvolution strategy, it is important to carefully select a strategy on the basis of the test compound characteristics.     

药物靶标蛋白在蛋白网络中的分布

药物靶标发现是目前生物学研究领域的热点和难点问题。从已有药物靶标中寻找规律可以为新靶标的发现总结规律,提供依据。随着功能基因组学的发展,这种组学数据的积累为这一问题的研究提供了契机。本文研究了已有靶标在蛋白网络中的分布,并分析了它们的蛋白功能域组成情况。结果显示靶标基因倾向位于网络的核心区域,并且集中在一些特定蛋白家族中。这些规律的总结将对药物研发过程中药物靶点的选择提供一定的帮助。     

Targeted protein degradation: Emerging concepts and protein state-specific targeting principles

Targeted protein degraders are heterobifunctional small molecules that link a target ligand or bait to an E3-ligase binder via a chemical spacer. Upon entering the cell, these ligands trigger the formation of a ternary complex between the target protein, degrader and E3-ligase, which leads to target polyubiquitination and proteasomal degradation. In recent years, TPD has expanded rapidly as a field, becoming the modality of choice in drug discovery and chemical probe development. This has been driven by the unique pharmacology of these molecules, which allows for fast and reversible knockdown of the target protein. Recent studies have demonstrated that degraders with specificity for a defined subpopulation of a protein-of-interest can be developed, giving rise to the emerging concept of protein state-specific targeting. In this article, we review advances towards developing degraders that differentiate between target protein subpopulations based on their; activation state, oligomerization state, cellular localization state, and cell type.     

Towards structure-based protein drug design

Structure-based drug design for chemical molecules has been widely used in drug discovery in the last 30 years. Many successful applications have been reported, especially in the field of virtual screening based on molecular docking. Recently, there has been much progress in fragment-based as well as de novo drug discovery. As many protein-protein interactions can be used as key targets for drug design, one of the solutions is to design protein drugs based directly on the protein complexes or the target structure. Compared with protein-ligand interactions, protein-protein interactions are more complicated and present more challenges for design. Over the last decade, both sampling efficiency and scoring accuracy of protein-protein docking have increased significantly. We have developed several strategies for structure-based protein drug design. A grafting strategy for key interaction residues has been developed and successfully applied in designing erythropoietin receptor-binding proteins. Similarly to small-molecule design, we also tested de novo protein-binder design and a virtual screen of protein binders using protein-protein docking calculations. In comparison with the development of structure-based small-molecule drug design, we believe that structure-based protein drug design has come of age.     

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.     

Modulating protein-protein interactions with small molecules: the importance of binding hotspots

The modulation of protein-protein interactions (PPIs) by small drug-like molecules is a relatively new area of research and has opened up new opportunities in drug discovery. However, the progress made in this area is limited to a handful of known cases of small molecules that target specific diseases. With the increasing availability of protein structure complexes, it is highly important to devise strategies exploiting homologous structure space on a large scale for discovering putative PPIs that could be attractive drug targets. Here, we propose a scheme that allows performing large-scale screening of all protein complexes and finding putative small-molecule and/or peptide binding sites overlapping with protein-protein binding sites (so-called "multibinding sites"). We find more than 600 nonredundant proteins from 60 protein families with multibinding sites. Moreover, we show that the multibinding sites are mostly observed in transient complexes, largely overlap with the binding hotspots and are more evolutionarily conserved than other interface sites. We investigate possible mechanisms of how small molecules may modulate protein-protein binding and discuss examples of new candidates for drug design.     

RNA as a target for small molecules

Proteins are folded to form a small binding site for catalysis or ligand recognition and this small binding site is traditionally the target for drug discovery. An alternative target for potential drug candidates is the translational process, which requires a precise reading of the entire mRNA sequence and, therefore, can be interrupted with small molecules that bind to mRNA sequence-specifically. RNA thus presents itself as a new upstream target for drug discovery because of the critical role it plays in the life of pathogens and in the progression of diseases. In this post-genomic era, RNA is becoming increasingly amenable to small-molecule therapy as greater structural and functional information accumulates with regard to important RNA functional domains. The study of aminoglycoside antibiotics and their binding to 16S ribosomal RNA has been a paradigm for our understanding of the ways in which small molecules can be developed to affect the function of RNA.     

The use of peptides in Diogenesis: a novel approach to drug discovery and phenomics

The Diogenesis Process is an integrated drug discovery platform that allows target validation, partner identification, and the identification of small molecule drug candidates for protein:protein interactions. Diogenesis utilizes the well-established methods of peptide display, synthetic and recombinant peptide production, in vitro biochemical and cell-based testing to form a universal drug discovery engine with distinct advantages over competing protocols. The process creates a library of diverse peptides, and selects rare and unique binders that identify and simplify surface "hot spots" on protein targets through which target activity can be regulated. In many cases, these peptide "Surrogates" have the minimal sequence and structural information needed to induce a change in the biological activity of the target; in pharmacological terms, only after inducing agonism or antagonism. The use of Surrogates in hot spot identification also allows subdivision of rather large surface domains into smaller domains that alone, or in combination with another subdomain, offers sufficient territory for modification of target activity. These Surrogates, in turn, provide the necessary ligands to develop appropriate Site Directed Assays (SDAs) for each essential subdomain. The SDAs provide the screening mode for finding competitive small molecules by high throughput screening. The other arm of the Diogenesis system is an application in the new area of "Phenomics." This part of the discovery process is a form of phenotypic analysis of genomic information that has also been referred to as "functional" genomics. Phenomics, done via the Diogenesis system, uses peptide Surrogates as modifiers of the activity of, and identifiers of the partners of, gene products of known and unknown function. Actually, in many instances, the same Surrogate isolated for use in Phenomics will be used to create SDAs for discovery of small molecule drug candidates. In this simple fashion, the two applications of Diogenesis are integrated to provide savings in research time and money.     

Analysis of multiple compound–protein interactions reveals novel bioactive molecules

The discovery of novel bioactive molecules advances our systems‐level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound–protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold‐hopping compounds. Through a machine‐learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G‐protein‐coupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand‐screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes.     

FtsZ inhibitors as a new genera of antibacterial agents

The continuous emergence and rapid spread of a multidrug-resistant strain of bacterial pathogens have demanded the discovery and development of new antibacterial agents. A highly conserved prokaryotic cell division protein FtsZ is considered as a promising target by inhibiting bacterial cytokinesis. Inhibition of FtsZ assembly restrains the cell-division complex known as divisome, which results in filamentation, leading to lysis of the cell. This review focuses on details relating to the structure, function, and influence of FtsZ in bacterial cytokinesis. It also summarizes on the recent perspective of the known natural and synthetic inhibitors directly acting on FtsZ protein, with prominent antibacterial activities. A series of benzamides, trisubstituted benzimidazoles, isoquinolene, guanine nucleotides, zantrins, carbonylpyridine, 4 and 5-Substituted 1-phenyl naphthalenes, sulindac, vanillin analogues were studied here and recognized as FtsZ inhibitors that act either by disturbing FtsZ polymerization and/or GTPase activity. Doxorubicin, from a U.S. FDA, approved drug library displayed strong interaction with FtsZ. Several of the molecules discussed, include the prodrugs of benzamide based compound PC190723 (TXA-709 and TXA707). These molecules have exhibited the most prominent antibacterial activity against several strains of Staphylococcus aureus with minimal toxicity and good pharmacokinetics properties. The evidence of research reports and patent documentations on FtsZ protein has disclosed distinct support in the field of antibacterial drug discovery. The pressing need and interest shall facilitate the discovery of novel clinical molecules targeting FtsZ in the upcoming days.     

Recent advances in target identification of bioactive natural products

Natural products are a tremendous source of tool discovery for basic science and drug discovery for clinical uses. In contrast to the large number of compounds isolated from nature, however, the number of compounds whose target molecules have been identified so far is fairly limited. Elucidation of the mechanism of how bioactive small molecules act in cells to induce biological activity (mode of action) is an attractive but challenging field of basic biology. At the same time, this is the major bottleneck for drug development of compounds identified in cell-based and phenotype-based screening. Although researchers’ experience and inspiration have been crucial for successful target identification, recent advancements in genomics, proteomics, and chemical genomics have made this challenging task possible in a systematic fashion.     

Chemogenomics: drug discovery's panacea?

Chemogenomics aims towards the systematic identification of small molecules that interact with the products of the genome and modulate their biological function. This Opinion article summarizes the different knowledge-based chemogenomics strategies that are followed and outlines the challenges and opportunities that will impact drug discovery. Chemogenomics aims towards the systematic identification of small molecules that interact with the products of the genome and modulate their biological function. While historically the approach is based on efforts that systematically explore target gene families like kinases, today additional knowledge-based systematization principles are followed within early drug discovery projects which aim to biologically validate the targets and to identify starting points for chemical lead optimization. While the expectations of chemogenomics are very high, the reality of drug discovery is quite sobering with very high project attrition rates. This article summarizes the different knowledge-based chemogenomics strategies that are followed and outlines the challenges and potential opportunities that will impact drug discovery.     

多肽噬菌体展示

噬菌体展示技术已被广泛地应用于生物学研究的各个方面.利用它可融合表达多肽、蛋白质结构域和蛋白质.尤其是多肽噬菌体展示,已被作为一种便利的研究工具去发现和研究那些与受体、酶、凝集素、抗体、核酸以及其他生物分子亲和的多肽配基和酶的底物专一性,该技术在药物的发现,疫苗的设计等医学领域也有着潜在的应用价值.     

Small molecule modulators of Wnt/β-catenin signaling

The Wnt signal transduction pathway is dysregulated in many highly prevalent diseases, including cancer. Unfortunately, drug discovery efforts have been hampered by the paucity of targets and drug-like lead molecules amenable to drug discovery. Recently, we reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling by a unique mechanism, though the target responsible remains unknown. We interrogated the mechanism and structure–activity relationships to understand drivers of potency and to assist target identification efforts. We found inhibition of Wnt signaling by Niclosamide appears unique among the structurally-related anthelmintic agents tested and found the potency and functional response was dependent on small changes in the chemical structure of Niclosamide. Overall, these findings support efforts to identify the target of Niclosamide inhibition of Wnt/β-catenin signaling and the discovery of potent and selective modulators to treat human disease.     

Screening for ligands using a generic and high-throughput light-scattering-based assay

Rapid identification of small molecules that interact with protein targets using a generic screening method greatly facilitates the development of therapeutic agents. The authors describe a novel method for performing homogeneous biophysical assays in a high-throughput format. The use of light scattering as a method to evaluate protein stability during thermal denaturation in a 384-well format yields a robust assay with a low frequency of false positives. This novel method leads to the identification of interacting small molecules without the addition of extraneous fluorescent probes. The analysis and interpretation of data is rapid, with sensitivity for protein stability comparable to differential scanning calorimetry. The authors propose potential uses in drug discovery, structural genomics, and functional genomics as a method to evaluate small-molecule interactions, identify natural cofactors that stabilize target proteins, and identify natural substrates and products for previously uncharacterized protein targets.