Predictive, non-GLP models of secondary pharmacodynamics: putting the best compounds forward

Secondary pharmacodynamic studies of new chemical entities (NCEs) play a critical role in support of efficient drug discovery. In an era in which speed and efficiency are the norm for pharmaceutical discovery, the need to identify NCEs with greater patient tolerability continues to increase. Early use of secondary pharmacodynamic models (in vivo and in vitro) provides the foundation for critical, early decisions regarding lead molecules. Scientifically robust, non-GLP (good laboratory practices) secondary pharmacodynamic studies can eliminate compounds or structural series with undesirable profiles early, and may prove useful in defining structure-activity relationships (SARs) with regards to off-target effects.     

Will natural products remain an important source of drug research for the future?

In the highly competitive environment of contemporary pharmaceutical research, natural products provide a unique element of molecular diversity and biological functionality which is indispensable for drug discovery. The emergence of strategies to deliver drug leads from natural products within the same time frame as synthetic chemical screening has eliminated a major limitation of the past. At a more functional level, the application of molecular genetics techniques has permitted the manipulation of biosynthetic pathways for the generation of novel chemical species as well as rendering hitherto uncultivatable microorganisms accessible for secondary metabolite generation. These developments augur well for an industry confronted with the challenge of finding lead compounds directed at the plethora of new targets arising from genomics projects. The exploitation of structural chemical databases comprising a wide variety of chemotypes, in conjunction with databases on target genes and proteins, will facilitate the creation of new chemical entities through computational molecular modelling for pharmacological evaluation.     

Development of a high-content screening assay panel to accelerate mechanism of action studies for oncology research

Efficient elucidation of the biological mechanism of action of novel compounds remains a major bottleneck in the drug discovery process. To address this need in the area of oncology, we report the development of a multiparametric high-content screening assay panel at the level of single cells to dramatically accelerate understanding the mechanism of action of cell growth-inhibiting compounds on a large scale. Our approach is based on measuring 10 established end points associated with mitochondrial apoptosis, cell cycle disruption, DNA damage, and cellular morphological changes in the same experiment, across three multiparametric assays. The data from all of the measurements taken together are expected to help increase our current understanding of target protein functions, constrain the list of possible targets for compounds identified using phenotypic screens, and identify off-target effects. We have also developed novel data visualization and phenotypic classification approaches for detailed interpretation of individual compound effects and navigation of large collections of multiparametric cellular responses. We expect this general approach to be valuable for drug discovery across multiple therapeutic areas.     

Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography-tandem mass spectrometry

A semi-automatic, high-throughput method has been developed to rapidly assess plasma protein binding of new chemical entities in drug discovery phase. New chemical entities are mixed with plasma and the unbound fractions are separated from the bound fraction by ultrafiltration in a 96-well filtrate assembly. The unbound fractions are then analyzed by fast liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample handling is automated by a robotic system. Employing a cocktail approach where multiple new chemical entities are allowed to bind to plasma proteins in the same well has further increased the throughput. We have validated the method with 12 commercially available compounds. The plasma protein binding data obtained by this method are comparable with the literature values. This method enables the determination of protein binding for 32 compounds in one single experiment instead of 1-2 compounds using the conventional methods.     

Evaluation of fluorescence- and mass spectrometry-based CYP inhibition assays for use in drug discovery

The potential for metabolism-related drug-drug interactions by new chemical entities is assessed by monitoring the impact of these compounds on cytochrome P450 (CYP) activity using well-characterized CYP substrates. The conventional gold standard approach for in vitro evaluation of CYP inhibitory potential uses pooled human liver microsomes (HLM) in conjunction with prototypical drug substrates, often quantified by LC-MS/MS. However, fluorescent CYP inhibition assays, which use recombinantly expressed CYPs and fluorogenic probe substrates, have been employed in early drug discovery to provide low-cost, high-throughput assessment of new chemical entities. Despite its greatly enhanced throughput, this approach has been met with mixed success in predicting the data obtained with the conventional gold standard approach (HLM+LC-MS). The authors find that the predictivity of fluorogenic assays for the major CYP isoforms 3A4 and 2D6 may depend on the quality of the test compounds. Although the structurally more optimized marketed drugs yielded acceptable correlations between the fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9 (r2 = 0.5-0.7; p < 0.005), preoptimization, early discovery compounds yielded poorer correlations (r2 < or = 0.2) for 2 of these major isoforms, CYPs 3A4 and 2D6. Potential reasons for the observed differences are discussed.     

Label-free optical biosensor: A tool for G protein-coupled receptors pharmacology profiling and inverse agonists identification

Current GPCR cell-based assays often rely on the measurement of a loaded fluorescent dye, fluorescently tagged targets, or the expression of a reporter. These manipulations may alter the cellular physiology of the target GPCR, and the measurements may be subject to off-target interference of compounds. Label-free optical biosensor-based technologies that provide a noninvasive methodology to study GPCR activation and signaling have been developed. These technologies enable the evaluation of drug effects on various GPCRs that couple to different signal transduction pathways using only one assay platform. This technology is highly sensitive and detects inverse agonism, therefore providing a convenient tool to study the pharmacology of drugs. Furthermore, its real-time kinetic measurements give researchers additional information about the biological responses induced by the drug. This assay platform when applied in early drug discovery efforts can provide valuable information on the mechanism of action and pharmacology profiles of drug candidates.     

斑马鱼在新药发现中的应用

在过去20年里,斑马鱼已成为一种重要的模式脊椎动物,在发育、遗传、免疫、肿瘤和毒理等诸多研究领域中被广泛应用。近年来,斑马鱼作为活体模型越来越多地应用于某些生物学过程的药物筛选。通过斑马鱼初步筛选,在药物研发初期可确定化合物的生物学活性、毒性以及副作用等。最近的研究还发现,斑马鱼不仅用于新药筛选,还可用于药物结构的优化。本文重点介绍斑马鱼在新药发现中的应用。     

Systematics-guided bioprospecting for bioactive microbial natural products

Advances in the taxonomic characterization of microorganisms have accelerated the rate at which new producers of natural products can be understood in relation to known organisms. Yet for many reasons, chemical efforts to characterize new compounds from new microbes have not kept pace with taxonomic advances. That there exists an ever-widening gap between the biological versus chemical characterization of new microorganisms creates tremendous opportunity for the discovery of novel natural products through the calculated selection and study of organisms from unique, untapped, ecological niches. A systematics-guided bioprospecting, including the construction of high quality libraries of marine microbes and their crude extracts, investigation of bioactive compounds, and increasing the active compounds by precision engineering, has become an efficient approach to drive drug leads discovery. This review outlines the recent advances in these issues and shares our experiences on anti-infectious drug discovery and improvement of avermectins production as well.     

Developing hybrid molecule therapeutics for diverse enzyme inhibitory action: Active role of coumarin-based structural leads in drug discovery

Hybrid drugs featuring two or more potentially bioactive pharmacophores have been recognized as advanced and superior chemical entities to simultaneously modulate multiple drug targets of multifactorial diseases, thus overcoming the severe side effects associated with a single drug molecule. The selection of these chemical moieties to produce hybrid structures with druggable properties is generally facilitated by the observed and/or anticipated synergistic pharmacological activities of the individual molecules. In this perspective, coumarin template has extensively been studied in pursuit of structurally diverse leads for drug development due to high affinity and specificity to different molecular targets. This review highlights the most commonly exploited approaches conceptualizing the design and construction of hybrid molecules by coupling two or more individual fragments with or without an appropriate linker. In addition to the design strategies, this review also summarizes and reflects on the therapeutic potential of these hybrid molecules for diverse enzyme inhibitory action as well as their observed structure-activity relationship (SAR). Several key features of the synthesized hybrid structures that assert a profound impact on the inhibitory function have also been discussed alongside computational investigations, inhibitor molecular diversity and selectivity toward multiple drug targets. Finally, these drug discovery and development efforts should serve as a handy reference aiming to provide a useful platform for the exploration of new coumarin-based compounds with enhanced enzyme inhibitory profile.     

High Content Analysis of Primary Macrophages Hosting Proliferating Leishmania Amastigotes: Application to Anti-leishmanial Drug Discovery

Background/Objectives

Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.

Methodology/Principal Findings

We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.

Conclusions/Significance

Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.     

Chemogenomics approaches to novel target discovery

Chemogenomics involves the combination of a compound's effect on biological targets together with modern genomics technologies. The merger of these two methodologies is creating a new way to screen for compound-target interactions, as well as map chemical and biological space in a parallel fashion. The challenge associated with mining complex databases has initiated the development of many novel in silico tools to profile and analyze data in a systematic way. The ability to analyze the combinatorial effects of chemical libraries on biological systems will aid the discovery of new therapeutic entities. Chemogenomics provides a tool for the rapid validation of novel targeted therapeutics, where a specific molecular target is modulated by a small molecule. Along with targeted therapies comes the ability to discovery pathway nodes where a single molecular target might be an essential component of more than one disease. Several disease areas will benefit directly from the chemogenomics approach, the most advanced being cancer. A genetic loss-of-function screen can be modulated in the presence of a compound to search for genes or pathways involved in the compound's activity. Several recent papers highlight how chemogenomics is changing with RNA interference-based screening and shaping the discovery of new targeted therapies. Together, chemical and RNA interference-based screens open the door for a new way to discovery disease-associated genes and novel targeted therapies.     

An unbiased evaluation of CK2 inhibitors by chemoproteomics: characterization of inhibitor effects on CK2 and identification of novel inhibitor targets

Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2alpha and CK2alpha' were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors.     

Chemical kinomics: a powerful strategy for target deconvolution

Kinomics is an emerging and promising approach for deciphering kinomes. Chemical kinomics is a discipline of chemical genomics that is also referred to as "chemogenomics", which is derived from chemistry and biology. Chemical kinomics has become a powerful approach to decipher complicated phosphorylation-based cellular signaling networks with the aid of small molecules that modulate kinase functions. Moreover, chemical kinomics has played a pivotal role in the field of kinase drug discovery as it enables identification of new molecular targets of small molecule kinase modulators and/or exploitation of novel functions of known kinases and has also provided novel chemical entities as hit/lead compounds. In this short review, contemporary chemical kinomics technologies such as activity-based protein profiling, T7 kinasetagged phages, kinobeads, three-hybrid systems, fluorescenttagged kinase binding assays, and chemical genomic profiling are discussed along with a novel allosteric Bcr-Abl kinase inhibitor (GNF-2/GNF-5) as a successful application of chemical kinomics approaches.     

应用化学基因组信息预测小分子化合物的潜在生物靶标的理论方法

在后基因组时代,化学基因组技术在药物作用靶点的确认、小分子化合物对通路的作用,以及小分子先导化合物的识别等方面都有着广泛的应用,为新药研发提供了新的技术方法。本文主要介绍了当前几种基于化学基因组信息来预测小分子化合物潜在生物靶标的理论方法（包括化学相似性搜索方法、反向分子对接方法、数据挖掘方法以及生物活性谱图分析方法）,并分析了这些方法的优缺点以及应用前景。     

Statistical decoding of potent pools based on chemical structure

Pooling experiments are used as a cost-effective approach for screening chemical compounds as part of the drug discovery process in pharmaceutical companies. When a biologically potent pool is found, the goal is to decode the pool, i.e., to determine which of the individual compounds are potent. We propose augmenting the data on pooled testing with information on the chemical structure of compounds in order to complete the decoding process. This proposal is based on the well-known relationship between biological potency of a compound and its chemical structure. Application to real data from a drug discovery process at GlaxoSmithKline reveals a 100% increase in hit rate, namely, the number of potent compounds identified divided by the number of tests required.     

Forward chemical genetics: progress and obstacles on the path to a new pharmacopoeia

Forward chemical genetics is a new method to systematize the discovery and use of small molecules as tools for basic biological research. This approach requires three basic components: a library of compounds; an assay, in which the library is screened for a cellular or organismal phenotype; and a method to trace an active compound to its biological target. Bioactive compounds have traditionally been isolated from natural product extracts, although 'diversity-oriented synthesis' and commercial compound collections are gaining in prominence. New techniques, such as image-based screening and the cytoblot method, have increased the throughput of phenotypic assays. Strategies are also being developed to streamline target identification using molecular biological approaches.     

Structure-based virtual screening of chemical libraries for drug discovery

One of the main goals in drug discovery is to identify new chemical entities that have a high likelihood of binding to the target protein to elicit the desired biological response. To this end, virtual screening is being increasingly used as a complement to high-throughput screening to improve the speed and efficiency of the drug discovery and development process. The availability of inexpensive high-performance computing platforms in recent years has transformed this field into one that is highly diverse and rapidly evolving, where large chemical databases have been successfully screened to identify hits for a wide range of targets such as Bcl-2 family proteins, G protein-coupled receptors, kinases, metalloproteins, nuclear hormone receptors, proteases and many more.