Protein-protein interactions as targets for small molecule drug discovery

Protein-protein interactions represent a highly populated class of targets for drug discovery. However, such systems present a number of unique challenges. This review presents an analysis of individual protein-protein interaction systems which have recently yielded success in discovering drug-like inhibitors. The structural characteristics of the protein binding sites and the attributes of the small molecule ligands are focused upon, in an attempt to derive commonly shared principles that may be of general usefulness in future drug discovery efforts within this target class.     

High throughput screening for small molecule enhancers of the interferon signaling pathway to drive next-generation antiviral drug discovery

Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z'>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-μM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit.     

Affinity characterization-mass spectrometry methodology for quantitative analyses of small molecule protein binding in solution

Affinity characterization by mass spectrometry (AC–MS) is a novel LC–MS methodology for quantitative determination of small molecule ligand binding to macromolecules. Its most distinguishing feature is the direct determination of all three concentration terms of the equilibrium binding equation, i.e., (M), (L), and (ML), which denote the macromolecule, ligand, and the corresponding complex, respectively. Although it is possible to obtain the dissociation constant from a single mixing experiment, saturation analyses are still valuable for assessing the overall binding phenomenon based on an established formalism. In addition to providing the prerequisite dissociation constant and binding stoichiometry, the technique also provides valuable information about the actual solubility of both macromolecule and ligand upon dilution and mixing in binding buffers. The dissociation constants and binding mode for interactions of DNA primase and thymidylate synthetase (TS) with high and low affinity small molecule ligands were obtained using the AC–MS method. The data were consistent with the expected affinity of TS for these ligands based on dissociation constants determined by alternative thermal-denaturation techniques: TdF or TdCD, and also consistent enzyme inhibition constants reported in the literature. The validity of AC–MS was likewise extended to a larger set of soluble protein–ligand systems. It was established as a valuable resource for counter screen and structure–activity relationship studies in drug discovery, especially when other classical techniques could only provide ambiguous results.     

Metagenomic small molecule discovery methods

1. Download : Download high-res image (213KB)
2. Download : Download full-size image

     

Miniaturized screening technologies for drug discovery

Adaptive Profiling (APL) and other biochip companies aim to harness the power of microsystems technology together with advances in chemistry and molecular biology, to become service and technology providers to organizations involved in pharmaceutical research and development. By supplying a unique range of decision-making tools that aid an earlier identification of qualified drug candidates for clinical development, the company should gain a significant share of the 10 billion US dollar biological screening, bioavailability and toxicity assessment market.     

NMR screening in drug discovery

NMR methods in drug discovery have traditionally been used to obtain structural information for drug targets or target-ligand complexes. Recently, it has been shown that NMR may be used as an alternative approach for identification of ligands that bind to protein drug targets, shifting the emphasis of many NMR laboratories towards screening and design of potential drug molecules, rather than structural characterization.     

Hypothesis testing in high-throughput screening for drug discovery

Following the success of small-molecule high-throughput screening (HTS) in drug discovery, other large-scale screening techniques are currently revolutionizing the biological sciences. Powerful new statistical tools have been developed to analyze the vast amounts of data in DNA chip studies, but have not yet found their way into compound screening. In HTS, characterization of single-point hit lists is often done only in retrospect after the results of confirmation experiments are available. However, for prioritization, for optimal use of resources, for quality control, and for comparison of screens it would be extremely valuable to predict the rates of false positives and false negatives directly from the primary screening results. Making full use of the available information about compounds and controls contained in HTS results and replicated pilot runs, the Z score and from it the p value can be estimated for each measurement. Based on this consideration, we have applied the concept of p-value distribution analysis (PVDA), which was originally developed for gene expression studies, to HTS data. PVDA allowed prediction of all relevant error rates as well as the rate of true inactives, and excellent agreement with confirmation experiments was found.     

Virtual screening strategies in drug discovery

The identification of novel therapeutic targets and characterization of their 3D structures is increasing at a dramatic rate. Computational screening methods continue to be developed and improved as credible and complementary alternatives to high-throughput biochemical compound screening (HTS). While the majority of drug candidates currently being developed have been found using HTS methods, high-throughput docking and pharmacophore-based searching algorithms are gaining acceptance and becoming a major source of lead molecules in drug discovery. Refinements and optimization of high-throughput docking methods have lead to improvements in reproducing experimental data and in hit rates obtained, validating their use in hit identification. In parallel with virtual screening methods, concomitant developments in cheminformatics including identification, design and manipulation of drug-like small molecule libraries have been achieved. Herein, currently used in silico screening techniques and their utility on a comparative and target dependent basis is discussed.     

虚拟筛选与新药发现

虚拟筛选是创新药物研究的新方法和新技术,近年来引起了研究机构和制药公司的高度重视,并且已经成为一种与高通量筛选互补的实用化工具,加入到了创新药物研究的工作流程(pipeline)中。本文介绍国际上虚拟筛选及其在创新药物发现中应用的研究进展,特别介绍了我国这方面研究的状况。     

Targeted 8-hydroxyquinoline fragment based small molecule drug discovery against neglected botulinum neurotoxin type F

ObjectivesBotulinum neurotoxins are highly potent biological warfare agents. The unavailability of countermeasures against these neurotoxins has been a matter of extensive research. However, no clinical therapeutics has come to existence till date. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored.MethodsIn present work, three course studies were performed involving in silico, in vitro and in vivo cascade to screen 8-HQ small molecule inhibitors against BoNT/F intoxication. ~800 molecules obtained from open repositories were screened in silico and commercially obtained twenty-four 8-HQ derived small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay. Selected compounds were further evaluated through endopeptidase assay. Further binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in vivo efficacy of these compounds was evaluated in mice model.ResultsThree compounds NSC1011, NSC1014 and NSC84094 were found to be highly inhibitory after screening of 8-HQ compounds through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed highest affinity binding of NSC1014 (K_D: 5.58E-06) with BoNT/F-LC. NSC1011, NSC1014, and NSC84094 displayed IC₅₀ of 30.47 ± 6.24, 14.91 ± 2.49 and 17.39 ± 2.74 μM, respectively, in endopeptidase assay. NSC1011 and NSC1014 displayed marked extension of survival time in mice model.ConclusionNSC1011 and NSC1014 have emerged as promising drug candidate against BoNT/F intoxication displaying higher potential than previously reported compounds.     

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.     

药物筛选新方法--高通量筛选

介绍了高通量筛选的概念、原理及其各个组成部分,着重阐明了高通量筛选的筛选模式,检测方法和应用实例,并简单介绍了国内在这方面的研究进展。     

BEAR, a novel virtual screening methodology for drug discovery

BEAR (binding estimation after refinement) is a new virtual screening technology based on the conformational refinement of docking poses through molecular dynamics and prediction of binding free energies using accurate scoring functions. Here, the authors report the results of an extensive benchmark of the BEAR performance in identifying a smaller subset of known inhibitors seeded in a large (1.5 million) database of compounds. BEAR performance proved strikingly better if compared with standard docking screening methods. The validations performed so far showed that BEAR is a reliable tool for drug discovery. It is fast, modular, and automated, and it can be applied to virtual screenings against any biological target with known structure and any database of compounds.     

Rational screening of oligonucleotide combinatorial libraries for drug discovery.

Combinatorial strategies offer the potential to generate and screen extremely large numbers of compounds and to identify individual molecules with a desired binding specificity or pharmacological activity. We describe a combinatorial strategy for oligonucleotides in which the library is generated and screened without using enzymes. Freedom from enzymes enables the use of oligonucleotide analogues. This dramatically extends the scope of both the compounds and the targets that may be screened. We demonstrate the utility of the method by screening 2'-O-Methyl and phosphorothioate oligonucleotide analogue libraries. Compounds have been identified that bind to the activated H-ras mRNA and that have potent antiviral activity against the human herpes simplex virus.     

A stem cell-based tool for small molecule screening in adipogenesis

Techniques for small molecule screening are widely used in biological mechanism study and drug discovery. Here, we reported a novel adipocyte differentiation assay for small molecule selection, based on human mesenchymal stem cells (hMSCs) transduced with fluorescence reporter gene driven by adipogenic specific promoter--adipocyte Protein 2 (aP2; also namely Fatty Acid Binding Protein 4, FABP4). During normal adipogenic induction as well as adipogenic inhibition by Ly294002, we confirmed that the intensity of green fluorescence protein corresponded well to the expression level of aP2 gene. Furthermore, this variation of green fluorescence protein intensity can be read simply through fluorescence spectrophotometer. By testing another two small molecules in adipogenesis--Troglitazone and CHIR99021, we proved that this is a simple and sensitive method, which could be applied in adipocyte biology, drug discovery and toxicological study in the future.     

Recent advances in small molecule drug delivery

The majority of new drugs, and new drug products, being developed and marketed by the pharmaceutical industry are small molecules. Oral administration remains the most common route of delivering such drugs, typically in the form of immediate-release tablets or capsules. While the immediate-release dosage forms dominate the market today, more specialized and rationalized products incorporating the concepts of drug delivery are being developed to overcome the physicochemical, physiological and pharmacological challenges inherent with the drugs, and to improve the treatment regimens for the patients. Today, these specialized concepts are increasingly being applied to first-generation products and not just products intended for the life cycle management of the franchise.     

High-throughput high-performance liquid chromatography/mass spectrometry for modern drug discovery

High-throughput high-performance liquid chromatography/mass spectrometry can be used in the analysis of high-throughput organic synthesis products, bioanalytical target analysis for preclinical and clinical studies, and early absorption, distribution, metabolism and excretion (ADME) screening. New techniques are emerging, including system automation, faster analysis, programmed multiple extraction and analysis columns, multiple electrospray ionization channels, and automated 96-well sample preparation.