Brent R. Stockwell

A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death

Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.

Privileged scaffolds for library design and drug discovery

This review explores the concept of using privileged scaffolds to identify biologically active compounds through building chemical libraries. We hope to accomplish three main objectives: to provide one of the most comprehensive listings of privileged scaffolds; to reveal through four selected examples the present state of the art in privileged scaffold library synthesis (in hopes of inspiring new and even more creative approaches); and also to offer some thoughts on how new privileged scaffolds might be identified and exploited.

Multicomponent therapeutics for networked systems

Therapeutic regimens that comprise more than one active ingredient are commonly used in clinical medicine. Despite this, most drug discovery efforts search for drugs that are composed of a single chemical entity. A focus in the early drug discovery process on identifying and optimizing the activity of combinations of molecules can result in the identification of more effective drug regimens. A systems perspective facilitates an understanding of the mechanism of action of such drug combinations.

Systematic discovery of multicomponent therapeutics

Multicomponent therapies, originating through deliberate mixing of drugs in a clinical setting, through happenstance, and through rational design, have a successful history in a number of areas of medicine, including cancer, infectious diseases, and CNS disorders. We have developed a high-throughput screening method for identifying effective combinations of therapeutic compounds. We report here that systematic screening of combinations of small molecules reveals unexpected interactions between compounds, presumably due to interactions between the pathways on which they act. Through systematic screening of ≈120,000 different two-component combinations of reference-listed drugs, we identified potential multicomponent therapeutics, including (i) fungistatic and analgesic agents that together generate fungicidal activity in drug-resistant Candida albicans, yet do not significantly affect human cells, (ii) glucocorticoid and antiplatelet agents that together suppress the production of tumor necrosis factor-α in human primary peripheral blood mononu-clear cells, and (iii) antipsychotic and antiprotozoal agents that do not exhibit significant antitumor activity alone, yet together prevent the growth of tumors in mice. Systematic combination screening may ultimately be useful for exploring the connectivity of biological pathways and, when performed with reference-listed drugs, may result in the discovery of new combination drug regimens.

Conserved pathways within bacteria and yeast as revealed by global protein network alignment

We implement a strategy for aligning two protein–protein interaction networks that combines interaction topology and protein sequence similarity to identify conserved interaction pathways and complexes. Using this approach we show that the protein–protein interaction networks of two distantly related species, Saccharomyces cerevisiae and Helicobacter pylori, harbor a large complement of evolutionarily conserved pathways, and that a large number of pathways appears to have duplicated and specialized within yeast. Analysis of these findings reveals many well characterized interaction pathways as well as many unanticipated pathways, the significance of which is reinforced by their presence in the networks of both species.

The role of iron and reactive oxygen species in cell death

The transition metal iron is essential for life, yet potentially toxic iron-catalyzed reactive oxygen species (ROS) are unavoidable in an oxygen-rich environment. Iron and ROS are increasingly recognized as important initiators and mediators of cell death in a variety of organisms and pathological situations. Here, we review recent discoveries regarding the mechanism by which iron and ROS participate in cell death. We describe the different roles of iron in triggering cell death, targets of iron-dependent ROS that mediate cell death and a new form of iron-dependent cell death termed ferroptosis. Recent advances in understanding the role of iron and ROS in cell death offer unexpected surprises and suggest new therapeutic avenues to treat cancer, organ damage and degenerative disease.

Regulation of ferroptotic cancer cell death by GPX4.

Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.

Exploring biology with small organic molecules

Small organic molecules have proven to be invaluable tools for investigating biological systems, but there is still much to learn from their use. To discover and to use more effectively new chemical tools to understand biology, strategies are needed that allow us to systematically explore ‘biological-activity space’. Such strategies involve analysing both protein binding of, and phenotypic responses to, small organic molecules. The mapping of biological-activity space using small molecules is akin to mapping the stars — uncharted territory is explored using a system of coordinates that describes where each new feature lies.

CHEMICAL GENETICS: LIGAND-BASED DISCOVERY OF GENE FUNCTION

Chemical genetics is the study of gene-product function in a cellular or organismal context using exogenous ligands. In this approach, small molecules that bind directly to proteins are used to alter protein function, enabling a kinetic analysis of the in vivo consequences of these changes. Recent advances have strongly enhanced the power of exogenous ligands such that they can resemble genetic mutations in terms of their general applicability and target specificity. The growing sophistication of this approach raises the possibility of its application to any biological process.Key PointsChemical genetics is the study of gene product function in a cellular or organismal context using exogenous ligands. The use of exogenous ligands requires three critical technologies: first, diverse chemical or peptide libraries; second, high-throughput screening; and last, protein target identification. To test the effects of the thousands or millions of compounds in large chemical-libraries, rapid methods are needed to conduct many assays in parallel. Target-based screens, which identify ligands for a specific protein of interest, are conceptually analogous to reverse-genetic methods, such as gene targeting in mice. Ligands to a specific protein target can be used to highlight the phenotypic consequences of inhibiting or otherwise altering the target protein.Phenotype-based screens test the ability of a peptide or small organic molecule to induce a specific phenotypic outcome in a cell or organism.DNA microarrays are useful for identifying the protein targets of small molecules.