Nicholas J. Westwood

Discovery, In Vivo Activity, and Mechanism of Action of a Small-Molecule p53 Activator

Summary We have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.

A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods

BackgroundCell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries.ResultsWe have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate.ConclusionsThe adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.

Isolation of Functionalized Phenolic Monomers through Selective Oxidation and CO Bond Cleavage of the β‐O‐4 Linkages in Lignin

Functionalized phenolic monomers have been generated and isolated from an organosolv lignin through a two-step depolymerization process. Chemoselective catalytic oxidation of β-O-4 linkages promoted by the DDQ/tBuONO/O2 system was achieved in model compounds, including polymeric models and in real lignin. The oxidized β-O-4 linkages were then cleaved on reaction with zinc. Compared to many existing methods, this protocol, which can be achieved in one pot, is highly selective, giving rise to a simple mixture of products that can be readily purified to give pure compounds. The functionality present in these products makes them potentially valuable building blocks.

Aromatic Monomers by in Situ Conversion of Reactive Intermediates in the Acid-Catalyzed Depolymerization of Lignin

Conversion of lignin into well-defined aromatic chemicals is a highly attractive goal but is often hampered by recondensation of the formed fragments, especially in acidolysis. Here, we describe new strategies that markedly suppress such undesired pathways to result in diverse aromatic compounds previously not systematically targeted from lignin. Model studies established that a catalytic amount of triflic acid is very effective in cleaving the β-O-4 linkage, most abundant in lignin. An aldehyde product was identified as the main cause of side reactions under cleavage conditions. Capturing this unstable compound by reaction with diols and by in situ catalytic hydrogenation or decarbonylation lead to three distinct groups of aromatic compounds in high yields acetals, ethanol and ethyl aromatics, and methyl aromatics. Notably, the same product groups were obtained when these approaches were successfully extended to lignin. In addition, the formation of higher molecular weight side products was markedly suppressed, indicating that the aldehyde intermediates play a significant role in these processes. The described strategy has the potential to be generally applicable for the production of interesting aromatic compounds from lignin.

Efficient Genome Engineering of Toxoplasma gondii Using CRISPR/Cas9

Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.

Acetylation site specificities of lysine deacetylase inhibitors in human cells

Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases.

Novel Cambinol Analogs as Sirtuin Inhibitors: Synthesis, Biological Evaluation, and Rationalization of Activity

The tenovins and cambinol are two classes of sirtuin inhibitor that exhibit antitumor activity in preclinical models. This report describes modifications to the core structure of cambinol, in particular by incorporation of substituents at the N1-position, which lead to increased potency and modified selectivity. These improvements have been rationalized using molecular modeling techniques. The expected functional selectivity in cells was also observed for both a SIRT1 and a SIRT2 selective analog.

Catalytic depolymerisation of isolated lignins to fine chemicals using a Pt/alumina catalyst: part 1—impact of the lignin structure

Four lignin preparations with different contents of alkyl–aryl ether bonds were depolymerised using an alumina supported platinum catalyst. The results showed that the proportion of β-O-4 linkages is the crucial factor for both the yield and the nature of the monomeric products. Highly condensed lignin generated mainly non-alkylated phenolic products while uncondensed lignin generated mainly phenolic products retaining the 3-carbon side-chain. These phenolic products with the 3-carbon chain still attached were considerably less abundant than the maximum potential yield calculated from selective cleavage of alkyl–aryl ether bonds by thioacidolysis, demonstrating that the scope for improved yield remains. Although the catalytic conversion yield rose with an increasing content of labile ether linkages in the lignin structure, optimisation of the catalytic depolymerisation was increasingly required to minimize side reactions. Gel permeation chromatography showed that the products converged towards the same molecular weight distribution regardless of the starting material. The full potential of the highly uncondensed lignin was reached only after the minimisation of condensation reactions during the catalytic conversion.

Pilot screening programme for small molecule activators of p53

Activation of the p53 tumour suppressor is predicted to have therapeutically beneficial effects. Many current anti‐cancer therapies activate the p53 response via DNA damage. Non‐genotoxic activation of the p53 pathway would open the way to long‐term and possibly prophylactic treatments. We have established a simple protocol to screen small compound libraries for activators of p53‐dependent transcription, and to select and characterise the most interesting hits, which include non‐genotoxic activators. These compounds or their derivatives are of potential clinical interest. This approach may also lead to the identification of novel p53‐activating compound families and possibly to the description of novel molecular pathways regulating p53 activity.

Using small molecules to study big questions in cellular microbiology

High‐throughput screening of small molecules is used extensively in pharmaceutical settings for the purpose of drug discovery. In the case of antimicrobials, this involves the identification of small molecules that are significantly more toxic to the microbe than to the host. Only a small percentage of the small molecules identified in these screens have been studied in sufficient detail to explain the molecular basis of their antimicrobial effect. Rarer still are small molecule screens undertaken with the explicit goal of learning more about the biology of a particular microbe or the mechanism of its interaction with its host. Recent technological advances in small molecule synthesis and high‐throughput screening have made such mechanism‐directed small molecule approaches a powerful and accessible experimental option. In this article, we provide an overview of the methods and technical requirements and we dis‐cuss the potential of small molecule approaches to address important and often otherwise experimentally intractable problems in cellular microbiology.