Systematic identification of proteins that elicit drug side effects

Side effect similarities of drugs have recently been employed to predict new drug targets, and networks of side effects and targets have been used to better understand the mechanism of action of drugs. Here, we report a large-scale analysis to systematically predict and characterize proteins that cause drug side effects. We integrated phenotypic data obtained during clinical trials with known drug–target relations to identify overrepresented protein–side effect combinations. Using independent data, we confirm that most of these overrepresentations point to proteins which, when perturbed, cause side effects. Of 1428 side effects studied, 732 were predicted to be predominantly caused by individual proteins, at least 137 of them backed by existing pharmacological or phenotypic data. We prove this concept in vivo by confirming our prediction that activation of the serotonin 7 receptor (HTR7) is responsible for hyperesthesia in mice, which, in turn, can be prevented by a drug that selectively inhibits HTR7. Taken together, we show that a large fraction of complex drug side effects are mediated by individual proteins and create a reference for such relations

High Concordance of Drug-Induced Human Hepatotoxicity and Other Target Organ Toxicity with in vitro Sublethal, Live-Cell Cytotoxicity Determined by High Content Screening

To develop and validate a practical, in vitro, cell-based model to assess human hepatotoxicity potential of drugs, we used the new technology of high content screening (HCS) and a novel combination of critical model features, including (1) use of live, human hepatocytes with drug metabolism capability, (2) preincubation of cells for 3 days with drugs at a range of concentrations up to at least 30 times the efficacious concentration or 100 uM, (3) measurement of multiple parameters that were (4) morphological and biochemical, (5) indicative of prelethal cytotoxic effects, (6) representative of different mechanisms of toxicity, (7) at the single cell level and (8) amenable to rapid throughput. HCS is based on automated epifluorescence microscopy and image analysis of cells in a microtiter plate format. The assay was applied to HepG2 human hepatocytes cultured in 96-well plates and loaded with four fluorescent dyes for: calcium (Fluo-4 AM), mitochondrial membrane potential (TMRM), DNA content (Hoechst 33342) to determine nuclear area and cell number and plasma membrane permeability (TOTO-3). Assay results were compared with those from 7 conventional, in vitro cytotoxicity assays that were applied to 611 compounds and shown to have low sensitivity (<25%), although high specificity (about 90%) for detection of toxic drugs. For 243 drugs with varying degrees of toxicity, the HCS, sublethal, cytotoxicity assay had a sensitivity of 93% and specificity of 98%. Drugs testing positive that did not cause hepatotoxicity produced other serious, human organ toxicities. For 201 positive assay results, 86% drugs affected cell number, 70% affected nuclear area and mitochondrial membrane potential and 45% affected membrane permeability and 41% intracellular calcium concentration. Cell number was the first parameter affected for 56% of these drugs, nuclear area for 34% and mitochondrial membrane potential for 29% and membrane permeability for 7% and intracellular calcium for 10%. Hormesis occurred for 48% of all drugs with positive response, for 26% of mitochondrial and 34% nuclear area changes and 12% of cell number changes. Pattern of change was dependent on the class of drug and mechanism of toxicity. The ratio of concentrations for in vitro cytotoxicity to maximal efficaciousness in humans was not different across groups (12\ub122). Human toxicity potential was detected with 80% sensitivity and 90% specificity at a concentration of 30x the maximal efficacious concentration or 100 uM when efficaciousness was not considered. We conclude that human hepatotoxicity is highly concordant with in vitro cytotoxicity in this novel model and as detected by HCS

In vitro assessment of a computer-designed potential anticancer agent in cervical cancer cells

BACKGROUND : Computer-based technology is becoming increasingly essential in biological research where drug discovery programs start with the identification of suitable drug targets. 2-Methoxyestradiol (2ME2) is a 17β-estradiol metabolite that induces apoptosis in various cancer cell lines including cervical cancer, breast cancer and multiple myeloma. Owing to 2ME2’s poor in vivo bioavailability, our laboratory in silico-designed and subsequently synthesized a novel 2ME2 analogue, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol), using receptor- and ligand molecular modeling. In this study, the biological effects of ESE-15-ol (180 nM) and its parent molecule, 2ME2 (1 μM), were assessed on morphology and apoptosis induction in cervical cancer cells. RESULTS : Transmission electron microscopy, scanning electron microscopy and polarization-optical transmitted light differential interference contrast (PlasDIC) images demonstrated morphological hallmarks of apoptosis including apoptotic bodies, shrunken cells, vacuoles, reduced cell density and cell debris. Flow cytometry analysis showed apoptosis induction by means of annexin V-FITC staining. Cell cycle analysis showed that ESE-15-ol exposure resulted in a statistically significant increase in the G2M phase (72%) compared to 2ME2 (19%). Apoptosis induction was more pronounced when cells were exposed to ESE-15-ol compared to 2ME2. Spectrophotometric analysis of caspase 8 activity demonstrated that 2ME2 and ESE-15-ol both induced caspase 8 activation by 2- and 1.7-fold respectively indicating the induction of the apoptosis. However, ESE-15-ol exerted all of the above-mentioned effects at a much lower pharmacological concentration (180 nM) compared to 2ME2 (1 μM physiological concentration). CONCLUSION : Computer-based technology is essential in drug discovery and together with in vitro studies for the evaluation of these in silico-designed compounds, drug development can be improved to be cost effective and time consuming. This study evaluated the anticancer potential of ESE-15-ol, an in silico-designed compound in vitro. Research demonstrated that ESE-15-ol exerts antiproliferative activity accompanied with apoptosis induction at a nanomolar concentration compared to the micromolar range required by 2ME2. This study is the first study to demonstrate the influence of ESE-15-ol on morphology, cell cycle progression and apoptosis induction in HeLa cells. In silico-design by means of receptor- and ligand molecular modeling is thus effective in improving compound bioavailability while preserving apoptotic activity in vitro.The Cancer Association of South Africa, the Struwig Germeshuysen Trust, RESCOM (School of Medicine, Research Committee of the Faculty of Health Sciences, University of Pretoria), the National Research Foundation and the Medical Research Council of South Africa.http://www.biolres.com/am2016Physiolog