Abstract: Background: Current drug discovery organizations have renewed interest in phenotypic/function based screening for the identification of novel small-molecule drug candidates. Phenotypic screening faces the challenge of deconvoluting the identity of molecular targets of small-molecules through which they exert their biological effect. The identity of the target is crucial for understanding the mechanism of drug action, rational drug design, interpretation of any toxicological findings and patient stratification. Several methods are available to deconvolute the targets of small-molecules. Objective: This review describes successful examples, limitations and advances of drug target deconvolution using small-molecule affinity chromatography coupled mass spectrometry based methods. A brief discussion of other target deconvolution methods is also presented for comparative appreciation of mass spectrometry based methods. Conclusion: The use of small-molecule affinity chromatography coupled mass spectrometry based...

Abstract: There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry-driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell-based phenotypic screening revealed that noncytotoxic concentrations of (Z)-(+/-)-2-(1-benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2. 2.2]octan-3-ol (analog I) and (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (analog II) inhibited endothelial cell migration and the ability to form capillary-like structures in Matrigel by > or =70%. The ability to undergo neoangiogenesis, as measured in a window-chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC(50) = 10 microM). Retroviral-mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small-molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation-mediated Caspase3-activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X-irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.

Abstract: Background: Phenotypic screening of random peptide libraries has been hampered by very poor hit rates. This is probably due to the fact that random combinatorial peptide libraries contain an insufficiently large proportion of secondary and/or tertiary structures that are likely to interact in a stable manner with multiple classes of potential target proteins. Phylomer libraries, by contrast, are derived from sequences of genomes that have been through millions to billions of years of evolution and were therefore hypothesized to be more likely to encode appropriate structures, which have been selected to stably bind with high affinity to protein surfaces. This approach is analogous to small-molecule libraries constructed to provide a rich source of structures (often found in natural products) that are common to the pharmacophores of known drugs. Discussion: Phenotypic screens of phylomer libraries show very high hit rates for bioactive peptides, suggesting that they may be a useful tool for target discovery and validation. Biophysical evidence suggests that this high activity may be due to the high proportion of affinities of unmodified peptides in the low nanomolar range. Conclusion: The high hit rates from phylomer libraries are sufficient to allow libraries composed of synthetic peptides to be synthesized and screened in parallel high-throughput screening formats. In addition to allowing the identification of new targets, the phylomer peptides themselves may be useful as structural probes to map epitopes of target vulnerability and as leads in therapeutic discovery.

Abstract: It is relatively simple and cheap for drug discovery by target-based screen in vitro, but the actions of drugs in vivo do not depend fully on the interactions between drugs and targets Major reasons preventing many early candidates reaching market are the inappropriate ADME (absorption, distribution, metabolism and excretion) properties and drug-induced toxicity Now more attentions were paid to the methods of drug screening in vivo In recent years, C elegans has been widely used as a drug screening model in drug discovery The developments of screening for drugs increasing lifespan and antagonizing microbes using C elegans-based assays were mainly discussed in this paper With the advantages of easily culture, simple tissue structure, and being amenable to high-throughput screening (HTS), C elegans may turn out to be invaluable in the development of novel screening methods in the future

Abstract: Background Praziquantel (PZQ) is the only widely available drug to treat schistosomiasis. Given the potential for drug resistance, it is prudent to search for novel therapeutics. Identification of anti-schistosomal chemicals has traditionally relied on phenotypic (whole organism) screening with adult worms in vitro and/or animal models of disease—tools that limit automation and throughput with modern microtiter plate-formatted compound libraries. Methods A partially automated, three-component phenotypic screen workflow is presented that utilizes at its apex the schistosomular stage of the parasite adapted to a 96-well plate format with a throughput of 640 compounds per month. Hits that arise are subsequently screened in vitro against adult parasites and finally for efficacy in a murine model of disease. Two GO/NO GO criteria filters in the workflow prioritize hit compounds for tests in the animal disease model in accordance with a target drug profile that demands short-course oral therapy. The screen workflow was inaugurated with 2,160 chemically diverse natural and synthetic compounds, of which 821 are drugs already approved for human use. This affords a unique starting point to ‘reposition’ (re-profile) drugs as anti-schistosomals with potential savings in development timelines and costs. Findings Multiple and dynamic phenotypes could be categorized for schistosomula and adults in vitro, and a diverse set of ‘hit’ drugs and chemistries were identified, including anti-schistosomals, anthelmintics, antibiotics, and neuromodulators. Of those hits prioritized for tests in the animal disease model, a number of leads were identified, one of which compares reasonably well with PZQ in significantly decreasing worm and egg burdens, and disease-associated pathology. Data arising from the three components of the screen are posted online as a community resource. Conclusions To accelerate the identification of novel anti-schistosomals, we have developed a partially automated screen workflow that interfaces schistosomula with microtiter plate-formatted compound libraries. The workflow has identified various compounds and drugs as hits in vitro and leads, with the prescribed oral efficacy, in vivo. Efforts to improve throughput, automation, and rigor of the screening workflow are ongoing.

Abstract: RNA interference (RNAi) mediated loss-of-function screens have the potential to delineate biological functions of genes and the proteins they encode. RNAi has proven to be a promising technology for identification and validation of new targets for the pharmacological treatment of many diseases including cancer. Here we review the use of high-throughput RNAi screens, examine the types of targets pursued for oncology indications, and discuss the integration of diverse datasets in both target discovery and drug discovery programs.

Abstract: Cell-based, phenotypic screening of small molecules often identifies compounds with provocative biological properties. However, determining the cellular target(s) and/or mechanism of action (MoA) of lead compounds remains an extremely challenging and time-consuming exercise. To provide insights into a compound's cellular action and greatly reduce the time required for MoA determination, we have developed a screening platform consisting of an extensive series of reporter gene assays (RGAs). A collection of > 11,000 compounds of known MoA (e.g., World Drug Index entries) were screened against the entire panel. The output provided evidence that an RGA signature could be ascribed to numerous, biologically diverse MoAs. The reference database generated suggested novel biological activity for particular compounds. For example, the profiling data led to the prediction that the cellular target of the natural product terprenin was dihydroorotate dehydrogenase (DHODH), which was confirmed experimentally. The screen...