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.     

Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization

Small molecule inhibitors of proteins are invaluable tools in research and as starting points for drug development. However, their screening can be tedious, as most screening methods have to be tailored to the corresponding drug target. Here, we describe a detailed protocol for a modular and generally applicable assay for the identification of small organic compounds that displace an aptamer complexed to its target protein. The method relies on fluorescence-labeled aptamers and the increase of fluorescence polarization upon their binding to the target protein. The assay has high Z'-factors, making it compatible with high-throughput screening. It allows easy automation, making fluorescence readout the time-limiting step. As aptamers can be generated for virtually any protein target, the assay allows identification of small molecule inhibitors for targets or individual protein domains for which no functional screen is available. We provide the step-by-step protocol to screen for antagonists of the cytohesin class of small guanosine exchange factors.     

Small molecule modulators of PCSK9 – A literature and patent overview

Proprotein convertase subtilisin kexin like type 9 (PCSK9) has since its discovery been a key protein target for the modulation of LDL cholesterol. The interest in PCSK9 has grown even more with the positive clinical trial outcomes in cardiovascular disease recently reported for two PCSK9 antibodies. Currently, there are no PCSK9 small molecule programs active in clinical development. However, there has been a steady increase in publications and patent applications within the PCSK9 small molecule field. This digest will provide a summary of small molecule and peptide PCSK9 modulators reported both in scientific journals and in patent applications, most of them originating from the last 3–4?years. As such, this digest will serve as an introduction to the field and assist further identification and discovery of small molecule PCSK9 modulators.     

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.     

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.     

Industrializing AI/ML during the end-to-end drug discovery process

Drug discovery aims to select proper targets and drug candidates to address unmet clinical needs. The end-to-end drug discovery process includes all stages of drug discovery from target identification to drug candidate selection. Recently, several artificial intelligence and machine learning (AI/ML)-based drug discovery companies have attempted to build data-driven platforms spanning the end-to-end drug discovery process. The ability to identify elusive targets essentially leads to the diversification of discovery pipelines, thereby increasing the ability to address unmet needs. Modern ML technologies are complementing traditional computer-aided drug discovery by accelerating candidate optimization in innovative ways. This review summarizes recent developments in AI/ML methods from target identification to molecule optimization, and concludes with an overview of current industrial trends in end-to-end AI/ML platforms.     

NMR techniques for characterization of ligand binding: utility for lead generation and optimization in drug discovery.

Over the last ten years, nmr spectroscopy has evolved into an important discipline in drug discovery. Initially, nmr was most useful as a technique to provide structural information regarding protein drug targets and target-ligand interactions. More recently, it has been shown that nmr may be used as an alternative method for identification of small molecule ligands that bind to protein drug targets. High throughput implementation of these experiments to screen small molecule libraries may lead to identification of potent and novel lead compounds. In this review, we will use examples from our own research to illustrate how nmr experiments to characterize ligand binding may be used to both screen for novel compounds during the process of lead generation, as well as provide structural information useful for lead optimization during the latter stages of a discovery program.     

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.     

Selection of bead‐displayed,PNA‐encoded chemicals

The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab‐on‐Bead? library, a one‐bead, one‐sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy‐aided identification of single target‐bound beads and the extraction – wet or dry – of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target‐binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target‐binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead‐based design allows for easy screening over a variety of prepared surfaces that can extend this technique's application to the discovery of diagnostic reagents and disease markers. Copyright © 2009 John Wiley & Sons, Ltd.     

基于亲和探针的药物靶点鉴定技术研究进展

药物靶点的鉴定和相关研究在药学研究领域具有重要的理论指导意义和实用价值。利用亲和探针偶联靶分子的方法是目前发现药 物靶点的主要手段之一。该方法可从分子水平发现药物的作用靶点,从而对药物的分子作用机制提供细胞水平的直接证据。从 DNA 和小 分子药物探针的构筑和应用入手,对近些年鉴定 DNA 损伤识别蛋白的研究进展进行了较为详尽的讨论,并简要介绍目前探索小分子药物 作用靶点的主流技术。作为亲和偶联鉴定药物作用靶点方法的重要组成部分,亲和探针设计的合理性关系到方法本身的可操作性以及鉴 定结果的可靠性。从多个角度对 DNA 探针和小分子药物探针的设计经验进行了较为系统的总结,例如经典的亲和纯化分离方法,以及更 为高效的光激发共价偶联技术等。这些方法和思路为探索 DNA 损伤相关蛋白质的功能以及小分子药物的细胞作用机制提供了丰富的研究 工具,有助于从分子水平理解药物的作用机制。     

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.     

Pharmacokinetic de-risking tools for selection of monoclonal antibody lead candidates

Pharmacokinetic studies play an important role in all stages of drug discovery and development. Recent advancements in the tools for discovery and optimization of therapeutic proteins have created an abundance of candidates that may fulfill target product profile criteria. Implementing a set of in silico, small scale in vitro and in vivo tools can help to identify a clinical lead molecule with promising properties at the early stages of drug discovery, thus reducing the labor and cost in advancing multiple candidates toward clinical development. In this review, we describe tools that should be considered during drug discovery, and discuss approaches that could be included in the pharmacokinetic screening part of the lead candidate generation process to de-risk unexpected pharmacokinetic behaviors of Fc-based therapeutic proteins, with an emphasis on monoclonal antibodies.     

Structural basis for CD44 recognition by ERM proteins

CD44 is an important adhesion molecule that functions as the major hyaluronan receptor which mediates cell adhesion and migration in a variety of physiological and pathological processes. Although full activity of CD44 requires binding to ERM (ezrin/radixin/moesin) proteins, the CD44 cytoplasmic region, consisting of 72 amino acid residues, lacks the Motif-1 consensus sequence for ERM binding found in intercellular adhesion molecule (ICAM)-2 and other adhesion molecules of the immunoglobulin superfamily. Ultracentrifugation sedimentation studies and circular dichroism measurements revealed an extended monomeric form of the cytoplasmic peptide in solution. The crystal structure of the radixin FERM domain complexed with a CD44 cytoplasmic peptide reveals that the KKKLVIN sequence of the peptide forms a beta strand followed by a short loop structure that binds subdomain C of the FERM domain. Like Motif-1 binding, the CD44 beta strand binds the shallow groove between strand beta5C and helix alpha1C and augments the beta sheet beta5C-beta7C from subdomain C. Two hydrophobic CD44 residues, Leu and Ile, are docked into a hydrophobic pocket with the formation of hydrogen bonds between Asn of the CD44 short loop and loop beta4C-beta5C from subdomain C. This binding mode resembles that of NEP (neutral endopeptidase 24.11) rather than ICAM-2. Our results reveal a characteristic versatility of peptide recognition by the FERM domains from ERM proteins, suggest a possible mechanism by which the CD44 tail is released from the cytoskeleton for nuclear translocation by regulated intramembrane proteolysis, and provide a structural basis for Smad1 interactions with activated CD44 bound to ERM protein.     

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。     

Fpocket: An open source platform for ligand pocket detection

Background  

Virtual screening methods start to be well established as effective approaches to identify hits, candidates and leads for drug discovery research. Among those, structure based virtual screening (SBVS) approaches aim at docking collections of small compounds in the target structure to identify potent compounds. For SBVS, the identification of candidate pockets in protein structures is a key feature, and the recent years have seen increasing interest in developing methods for pocket and cavity detection on protein surfaces.     

The role of fluorine in medicinal chemistry

The small and highly electronegative fluorine atom can play a remarkable role in medicinal chemistry. Selective installation of fluorine into a therapeutic or diagnostic small molecule candidate can enhance a number of pharmacokinetic and physicochemical properties such as improved metabolic stability and enhanced membrane permeation. Increased binding affinity of fluorinated drug candidates to target protein has also been documented in a number of cases. A further emerging application of the fluorine atom is the use of 18F as a radiolabel tracer atom in the exquisitely sensitive technique of Positron Emission Tomography (PET) imaging. This short review aims to bring together these various aspects of the use of fluorine in medicinal chemistry applications, citing selected examples from across a variety of therapeutic and diagnostic settings. The increasingly routine incorporation of fluorine atom(s) into drug candidates suggests a bright future for fluorine in drug discovery and development. A major challenge moving forward will be how and where to install fluorine in a rational sense to best optimise molecular properties.     

Chemical proteomics and its impact on the drug discovery process

Despite the rapid growth of postgenomic data and fast-paced technology advancement, drug discovery is still a lengthy and difficult process. More effective drug design requires a better understanding of the interaction between drug candidates and their targets/off-targets in various situations. The ability of chemical proteomics to integrate a multiplicity of disciplines enables the direct analysis of protein activities on a proteome-wide scale, which has enormous potential to facilitate drug target elucidation and lead drug verification. Over recent years, chemical proteomics has experienced rapid growth and provided a valuable method for drug target identification and inhibitor discovery. This review introduces basic concepts and technologies of different popular chemical proteomic approaches. It also covers the essential features and recent advances of each approach while underscoring their potentials in drug discovery and development.     

Drug discovery in academia

Drug discovery and development is generally done in the commercial rather than the academic realm. Drug discovery involves target discovery and validation, lead identification by high-throughput screening, and lead optimization by medicinal chemistry. Follow-up preclinical evaluation includes analysis in animal models of compound efficacy and pharmacology (ADME: administration, distribution, metabolism, elimination) and studies of toxicology, specificity, and drug interactions. Notwithstanding the high-cost, labor-intensive, and non-hypothesis-driven aspects of drug discovery, the academic setting has a unique and expanding niche in this important area of investigation. For example, academic drug discovery can focus on targets of limited commercial value, such as third-world and rare diseases, and on the development of research reagents such as high-affinity inhibitors for pharmacological "gene knockout" in animal models ("chemical genetics"). This review describes the practical aspects of the preclinical drug discovery process for academic investigators. The discovery of small molecule inhibitors and activators of the cystic fibrosis transmembrane conductance regulator is presented as an example of an academic drug discovery program that has yielded new compounds for physiology research and clinical development. high-throughput screening; drug development; pharmacology; fluorescence; cystic fibrosis transmembrane conductance regulator     

Improving success rates for lead generation using affinity binding technologies

Affinity technologies have been applied at several stages of the drug discovery process, ranging from target identification and purification to the identification of preclinical candidates. The detection of ligand-macromolecule interactions in lead discovery is the best studied and most powerful of these techniques. Although affinity methods have been in widespread use for about a decade, only recently have many reports emerged on their utility. Primary affinity screens of large libraries of small molecules or fragments have begun to produce results for challenging targets. Furthermore, in secondary assays affinity methods are opening new avenues to tackle important medicinal chemistry tasks.