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Novel classes of broad-spectrum antibiotics have been extremely difficult to discover, largely due to the impermeability of the Gram-negative membranes coupled with a poor understanding of the physicochemical properties a compound should possess to promote its accumulation inside the cell. To address this challenge, numerous methodologies for assessing intracellular compound accumulation in Gram-negative bacteria have been established, including classic radiometric and fluorescence-based methods. The recent development of accumulation assays that utilize liquid chromatography–tandem mass spectrometry (LC-MS/MS) have circumvented the requirement for labeled compounds, enabling assessment of a substantially broader range of small molecules. Our unbiased study of accumulation trends in Escherichia coli using an LC-MS/MS-based assay led to the development of the eNTRy rules, which stipulate that a compound is most likely to accumulate in E. coli if it has an ionizable Nitrogen, has low Three-dimensionality and is relatively Rigid. To aid in the implementation of the eNTRy rules, we developed a complementary web tool, eNTRyway, which calculates relevant properties and predicts compound accumulation. Here we provide a comprehensive protocol for analysis and prediction of intracellular accumulation of small molecules in E. coli using an LC-MS/MS-based assay (which takes ~2 d) and eNTRyway, a workflow that is readily adoptable by any microbiology, biochemistry or chemical biology laboratory. This protocol describes an LC-MS/MS assay and complementary web tool for analysis and prediction of intracellular accumulation of small molecules in E. coli based on their physiochemical properties, which could aid in future antibiotic discovery.

Most small molecules are unable to rapidly traverse the outer membrane of Gram-negative bacteria and accumulate inside these cells, making the discovery of much-needed drugs against these pathogens challenging. Current understanding of the physicochemical properties that dictate small-molecule accumulation in Gram-negative bacteria is largely based on retrospective analyses of antibacterial agents, which suggest that polarity and molecular weight are key factors. Here we assess the ability of over 180 diverse compounds to accumulate in Escherichia coli. Computational analysis of the results reveals major differences from the retrospective studies, namely that the small molecules that are most likely to accumulate contain an amine, are amphiphilic and rigid, and have low globularity. These guidelines were then applied to convert deoxynybomycin, a natural product that is active only against Gram-positive organisms, into an antibiotic with activity against a diverse panel of multi-drug-resistant Gram-negative pathogens. We anticipate that these findings will aid in the discovery and development of antibiotics against Gram-negative bacteria.

Natural products often serve as sources of new drugs, either directly or after synthetic modification, but site-selective functionalization of complex small molecules is challenging. Now, a method has been developed that enables selective modification of a wide range of natural products by engaging a benzyne intermediate in a variety of reaction modes.

High-throughput screening is the dominant method used to identify lead compounds in drug discovery. As such, the makeup of screening libraries largely dictates the biological targets that can be modulated and the therapeutics that can be developed. Unfortunately, most compound-screening collections consist principally of planar molecules with little structural or stereochemical complexity, compounds that do not offer the arrangement of chemical functionality necessary for the modulation of many drug targets. Here we describe a novel, general and facile strategy for the creation of diverse compounds with high structural and stereochemical complexity using readily available natural products as synthetic starting points. We show through the evaluation of chemical properties (which include fraction of sp(3) carbons, ClogP and the number of stereogenic centres) that these compounds are significantly more complex and diverse than those in standard screening collections, and we give guidelines for the application of this strategy to any suitable natural product.

PAC-1 is a preferential small molecule activator of procaspase-3 and has potential to become a novel and effective anticancer agent. The rational development of PAC-1 for translational oncologic applications would be advanced by coupling relevant in vitro cytotoxicity studies with pharmacokinetic investigations conducted in large mammalian models possessing similar metabolism and physiology as people. In the present study, we investigated whether concentrations and exposure durations of PAC-1 that induce cytotoxicity in lymphoma cell lines in vitro can be achievable in healthy dogs through a constant rate infusion (CRI) intravenous delivery strategy. Time- and dose-dependent procaspase-3 activation by PAC-1 with subsequent cytotoxicity was determined in a panel of B-cell lymphoma cells in vitro. The pharmacokinetics of PAC-1 administered orally or intravenously was studied in 6 healthy dogs using a crossover design. The feasibility of maintaining steady state plasma concentration of PAC-1 for 24 or 48 h that paralleled in vitro cytotoxic concentrations was investigated in 4 healthy dogs. In vitro, PAC-1 induced apoptosis in lymphoma cell lines in a time- and dose-dependent manner. The oral bioavailability of PAC-1 was relatively low and highly variable (17.8 ± 9.5%). The achievement and maintenance of predicted PAC-1 cytotoxic concentrations in normal dogs was safely attained via intravenous CRI lasting for 24 or 48 h in duration. Using the dog as a large mammalian model, PAC-1 can be safely administered as an intravenous CRI while achieving predicted in vitro cytotoxic concentrations.

Cribrostatin 6 is a quinone-containing natural product that induces the death of cancer cell lines in culture, and its mechanism of action and scope of activity are unknown. Here we show that cribrostatin 6 has broad anticancer activity, potently inducing apoptotic cell death that is not preceded by any defined cell cycle arrest. Consistent with this data, we find that cribrostatin 6 treated cells have large amounts of reactive oxygen species (ROS) and, based on transcript profiling experiments and other data, this ROS generation is likely the primary mechanism by which cribrostatin 6 induces apoptosis. Given the success of certain ROS producers as anticancer agents, cribrostatin 6 has potential as a novel chemotherapeutic agent.

This protocol describes the gram-scale solution-phase synthesis of the colorimetric caspase-3/7 substrate Ac-DEVD-pNA. The caspase enzymes are integral to cellular inflammation and apoptotic cascades, and are commonly studied by cell biologists, medicinal chemists and chemical biologists. In particular, the assessment of caspase enzymatic activity is a standard method to evaluate cell death pathways and new apoptosis-modulating agents. Caspase enzymatic activity can be conveniently monitored with peptidic chromogenic or fluorogenic substrates, with certain peptide sequences imparting selectivity for certain caspases. The synthesis of these peptide substrates is typically carried out by solid-phase synthesis, a method that is not ideal for production of the gram quantities needed for high-throughput screening. Described herein is a facile method for the synthesis of the Ac-DEVD-pNA caspase-3/7 substrate using solution-phase peptide synthesis. This protocol, involving iterative PyBOP-mediated couplings and Fmoc deprotections, is rapid (about 5 d), operationally simple and can be used to generate over 1 g of product at a fraction of the cost of the commercial substrate.

Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DNA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1ĝ€2ĝ€ 2-2ĝ€2) riboseĝ€"ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodanine-containing mammalian PARG inhibitor RBPI-3. Our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors. © 2012 Macmillan Publishers Limited. All rights reserved.