Bridget K. Wagner

Target identification and mechanism of action in chemical biology and drug discovery

Target-identification and mechanism-of-action studies have important roles in small-molecule probe and drug discovery. Biological and technological advances have resulted in the increasing use of cell-based assays to discover new biologically active small molecules. Such studies allow small-molecule action to be tested in a more disease-relevant setting at the outset, but they require follow-up studies to determine the precise protein target or targets responsible for the observed phenotype. Target identification can be approached by direct biochemical methods, genetic interactions or computational inference. In many cases, however, combinations of approaches may be required to fully characterize on-target and off-target effects and to understand mechanisms of small-molecule action.

Small molecules of different origins have distinct distributions of structural complexity that correlate with protein-binding profiles

Using a diverse collection of small molecules generated from a variety of sources, we measured protein-binding activities of each individual compound against each of 100 diverse (sequence-unrelated) proteins using small-molecule microarrays. We also analyzed structural features, including complexity, of the small molecules. We found that compounds from different sources (commercial, academic, natural) have different protein-binding behaviors and that these behaviors correlate with general trends in stereochemical and shape descriptors for these compound collections. Increasing the content of sp3-hybridized and stereogenic atoms relative to compounds from commercial sources, which comprise the majority of current screening collections, improved binding selectivity and frequency. The results suggest structural features that synthetic chemists can target when synthesizing screening collections for biological discovery. Because binding proteins selectively can be a key feature of high-value probes and drugs, synthesizing compounds having features identified in this study may result in improved performance of screening collections.

Large-scale chemical dissection of mitochondrial function

Mitochondrial oxidative phosphorylation (OXPHOS) is under the control of both mitochondrial (mtDNA) and nuclear genomes and is central to energy homeostasis. To investigate how its function and regulation are integrated within cells, we systematically combined four cell-based assays of OXPHOS physiology with multiplexed measurements of nuclear and mtDNA gene expression across 2,490 small-molecule perturbations in cultured muscle. Mining the resulting compendium revealed, first, that protein synthesis inhibitors can decouple coordination of nuclear and mtDNA transcription; second, that a subset of HMG-CoA reductase inhibitors, combined with propranolol, can cause mitochondrial toxicity, yielding potential clues about the etiology of statin myopathy; and, third, that structurally diverse microtubule inhibitors stimulate OXPHOS transcription while suppressing reactive oxygen species, via a transcriptional mechanism involving PGC-1α and ERRα, and thus may be useful in treating age-associated degenerative disorders. Our screening compendium can be used as a discovery tool both for understanding mitochondrial biology and toxicity and for identifying novel therapeutics.

Targeting the pancreatic β-cell to treat diabetes

Diabetes is a leading cause of morbidity and mortality worldwide, and predicted to affect over 500 million people by 2030. However, this growing burden of disease has not been met with a comparable expansion in therapeutic options. The appreciation of the pancreatic β-cell as a central player in the pathogenesis of both type 1 and type 2 diabetes has renewed focus on ways to improve glucose homeostasis by preserving, expanding and improving the function of this key cell type. Here, we provide an overview of the latest developments in this field, with an emphasis on the most promising strategies identified to date for treating diabetes by targeting the β-cell.

Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs.

Piperlongumine is a naturally occurring small molecule recently identified to be toxic selectively to cancer cells in vitro and in vivo. This compound was found to elevate cellular levels of reactive oxygen species (ROS) selectively in cancer cell lines. The synthesis of 80 piperlongumine analogs has revealed structural modifications that retain, enhance, and ablate key piperlongumine-associated effects on cells, including elevation of ROS, cancer cell death, and selectivity for cancer cells over nontransformed cell types. Structure/activity relationships suggest that the electrophilicity of the C2-C3 olefin is critical for the observed effects on cells. Furthermore, we show that analogs lacking a reactive C7-C8 olefin can elevate ROS to levels observed with piperlongumine but show markedly reduced cell death, suggesting that ROS-independent mechanisms, including cellular cross-linking events, may also contribute to piperlongumine’s induction of apoptosis. In particular, we have identified irreversible protein glutathionylation as a process associated with cellular toxicity. We propose a mechanism of action for piperlongumine that may be relevant to other small molecules having two sites of reactivity, one with greater and the other with lesser electrophilicity.

A small-molecule probe of the histone methyltransferase G9a induces cellular senescence in pancreatic adenocarcinoma.

Post-translational modifications of histones alter chromatin structure and play key roles in gene expression and specification of cell states. Small molecules that target chromatin-modifying enzymes selectively are useful as probes and have promise as therapeutics, although very few are currently available. G9a (also named euchromatin histone methyltransferase 2 (EHMT2)) catalyzes methylation of lysine 9 on histone H3 (H3K9), a modification linked to aberrant silencing of tumor-suppressor genes, among others. Here, we report the discovery of a novel histone methyltransferase inhibitor, BRD4770. This compound reduced cellular levels of di- and trimethylated H3K9 without inducing apoptosis, induced senescence, and inhibited both anchorage-dependent and -independent proliferation in the pancreatic cancer cell line PANC-1. ATM-pathway activation, caused by either genetic or small-molecule inhibition of G9a, may mediate BRD4770-induced cell senescence. BRD4770 may be a useful tool to study G9a and its role in senescence and cancer cell biology.

Correlating chemical sensitivity and basal gene expression reveals mechanism of action

Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with ~19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters, and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.

Gene expression-based screening identifies microtubule inhibitors as inducers of PGC-1α and oxidative phosphorylation

The transcriptional coactivator PGC-1α is a potent regulator of several metabolic pathways, including, in particular, the activation of oxidative phosphorylation and mitochondrial biogenesis. Recent evidence suggests that increasing PGC-1α activity may have beneficial effects in various conditions, including muscular dystrophy, diabetes, and neurodegenerative diseases. We describe here a high-throughput screen to identify small molecules that induce PGC-1α expression in skeletal muscle cells. A number of drug classes are identified, including glucocorticoids, microtubule inhibitors, and protein synthesis inhibitors. These drugs induce PGC-1α mRNA, and the expression of a number of genes known to be regulated by PGC-1α. No induction of these target genes is seen in PGC-1α −/− cells, demonstrating that the drugs act through PGC-1α. These data demonstrate the feasibility of high-throughput screening for inducers of PGC-1α. Moreover, the data identify microtubule inhibitors and protein synthesis inhibitors as modulators of PGC-1α and oxidative phosphorylation.

Towards patient-based cancer therapeutics

A new approach to the discovery of cancer therapeutics is emerging that begins with the cancer patient. Genomic analysis of primary tumors is providing an unprecedented molecular characterization of the disease. The next step requires relating the genetic features of cancers to acquired gene and pathway dependencies and identifying small-molecule therapeutics that target them.

Small-molecule inducers of insulin expression in pancreatic α-cells

High-content screening for small-molecule inducers of insulin expression identified the compound BRD7389, which caused α-cells to adopt several morphological and gene expression features of a β-cell state. Assay-performance profile analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a β-cell-like state. BRD7389 also increases the endocrine cell content and function of donor human pancreatic islets in culture.