Kenichi Shimada | Harvard Medical School (original) (raw)
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Papers by Kenichi Shimada
Cell chemical biology, Jan 7, 2018
Transition metals are essential, but deregulation of their metabolism causes toxicity. Here, we r... more Transition metals are essential, but deregulation of their metabolism causes toxicity. Here, we report that the compound NSC319726 binds copper to induce oxidative stress and arrest glioblastoma-patient-derived cells at picomolar concentrations. Pharmacogenomic analysis suggested that NSC319726 and 65 other structural analogs exhibit lethality through metal binding. Although NSC319726 has been reported to function as a zinc ionophore, we report here that this compound binds to copper to arrest cell growth. We generated and validated pharmacogenomic predictions: copper toxicity was substantially inhibited by hypoxia, through an hypoxia-inducible-factor-1α-dependent pathway; copper-bound NSC319726 induced the generation of reactive oxygen species and depletion of deoxyribosyl purines, resulting in cell-cycle arrest. These results suggest that metal-induced DNA damage may be a consequence of exposure to some xenobiotics, therapeutic agents, as well as other causes of copper dysregulati...
Nature chemical biology, 2014
We previously discovered a small-molecule inducer of cell death, named 1541, that noncovalently s... more We previously discovered a small-molecule inducer of cell death, named 1541, that noncovalently self-assembles into chemical fibrils ('chemi-fibrils') and activates procaspase-3 in vitro. We report here that 1541-induced cell death is caused by the fibrillar rather than the soluble form of the drug. A short hairpin RNA screen reveals that knockdown of genes involved in endocytosis, vesicle trafficking and lysosomal acidification causes partial 1541 resistance. We confirm the role of these pathways using pharmacological inhibitors. Microscopy shows that the fluorescent chemi-fibrils accumulate in punctae inside cells that partially colocalize with lysosomes. Notably, the chemi-fibrils bind and induce liposome leakage in vitro, suggesting they may do the same in cells. The chemi-fibrils induce extensive proteolysis including caspase substrates, yet modulatory profiling reveals that chemi-fibrils form a distinct class from existing inducers of cell death. The chemi-fibrils shar...
Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recogni... more Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.
Precision medicine in oncology requires not only identification of cancer-associated mutations bu... more Precision medicine in oncology requires not only identification of cancer-associated mutations but also effective drugs for each cancer genotype, which is still a largely unsolved problem. One approach for the latter challenge has been large-scale testing of small molecules in genetically characterized cell lines. We hypothesized that compounds with high cell-line-selective lethality exhibited consistent results across such pharmacogenomic studies. We analyzed the compound sensitivity data of 6,259 lethal compounds from the NCI-60 project. A total of 2,565 cell-line-selective lethal compounds were identified and grouped into 18 clusters based on their median growth inhibitory GI50 profiles across the 60 cell lines, which were shown to represent distinct mechanisms of action. Further transcriptome analysis revealed a biomarker, NADPH abundance, for predicting sensitivity to ferroptosis-inducing compounds, which we experimentally validated. In summary, incorporating cell-line-selectivity filters improves the predictive power of pharmacogenomic analyses and enables discovery of biomarkers that predict the sensitivity of cells to specific cell death inducers.
Cell, 2014
Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We soug... more Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosisinducing 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.
ACS Medicinal Chemistry Letters, 2012
We analyzed more than 1 million small molecules with the goal of finding simple synthetic compoun... more We analyzed more than 1 million small molecules with the goal of finding simple synthetic compounds that potently inhibit cancer cell growth. We identified three such compounds with unknown mechanisms of action. Subsequent studies revealed that all three of these small molecules target microtubules. These three scaffolds can serve as templates for developing new microtubule-targeted agents, overcoming the limits of existing microtubule-inhibiting drugs derived from complex natural products.
Journal of the American Chemical Society, 2014
Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, 18 nonapoptotic cell ... more Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, 18 nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of 19 Huntington's disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; 20 Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species 21 formation or lysosomal membrane permeability. We developed a mechanistic model to 22 explain the activity of Fer-1, which guided the development of ferrostatins with improved 23 properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid 24 peroxidation mediates diverse disease phenotypes.
Cancer discovery, 2011
E3 ubiquitin ligases are of interest as drug targets for their ability to regulate protein stabil... more E3 ubiquitin ligases are of interest as drug targets for their ability to regulate protein stability and function. The oncogene Mdm2 is an attractive E3 ligase to target, as it is the key negative regulator of the tumor suppressor p53, which controls the transcription of genes involved in cell fate. Overexpression of Mdm2 facilitates tumorigenesis by inactivating p53, and through p53-independent oncogenic effects. We developed a high-throughput cellular Mdm2 auto-ubiquitination assay, which we used to discover a class of small-molecule Mdm2 ligase activity inhibitors. These compounds inhibit Mdm2 and p53 ubiquitination in cells, reduce viability of cells with wild-type p53, and synergize with DNA-damaging agents to cause cell death. We determined that these compounds effectively inhibit the E3 ligase activity of the Mdm2-MdmX hetero-complex. This mechanism may be exploitable to create a new class of anti-tumor agents.
A series of Pictet-Spengler condensation derivatives (tetrahydro-b-carbolines) was designed, synt... more A series of Pictet-Spengler condensation derivatives (tetrahydro-b-carbolines) was designed, synthesized and evaluated for lethality against a panel of seven cancer cell lines. Seven compounds (2a, 13, 20, 21, 27, 29 and 34) showed lethality in at least five cell lines. Among these, compound 27 showed a unique selectivity towards oncogenic-RAS expressing BJ-TERT/LT/ST/RAS V12 tumor cells, compared to non-transformed BJ-TERT cells. Further investigation revealed that 27 induces cell death without activation of caspases. This represents a useful new probe of non-apoptotic cell death and oncogenic-RAS synthetic lethality.
Cell death is a complex process that plays a vital role in development, homeostasis, and disease.... more Cell death is a complex process that plays a vital role in development, homeostasis, and disease. Our understanding of and ability to control cell death is impeded by an incomplete characterization of the full range of cell death processes that occur in mammalian systems, especially in response to exogenous perturbations. We present here a general approach to address this problem, which we call modulatory profiling. Modulatory profiles are composed of the changes in potency and efficacy of lethal compounds produced by a second cell death-modulating agent in human cell lines. We show that compounds with the same characterized mechanism of action have similar modulatory profiles. Furthermore, clustering of modulatory profiles revealed relationships not evident when clustering lethal compounds based on gene expression profiles alone. Finally, modulatory profiling of compounds correctly predicted three previously uncharacterized compounds to be microtubule-destabilizing agents, classified numerous compounds that act nonspecifically, and identified compounds that cause cell death through a mechanism that is morphologically and biochemically distinct from previously established ones. apoptosis | chemical biology | small molecules C ell death has historically been viewed as a binary phenomenon. Cells were described to die in one of two ways-through a controlled and ordered process (apoptosis) or an unregulated and chaotic process (necrosis) (1, 2). Not only were these often considered the only two possible mechanisms, but they were also frequently viewed as morphologically and biochemically uniform (3, 4). A great deal of research in recent decades has not only shown the complexity and heterogeneity of apoptotic and necrotic signaling, but also that cells can die in physiological and nonphysiological contexts through processes morphologically and biochemically distinct from both apoptosis and necrosis.
Cell chemical biology, Jan 7, 2018
Transition metals are essential, but deregulation of their metabolism causes toxicity. Here, we r... more Transition metals are essential, but deregulation of their metabolism causes toxicity. Here, we report that the compound NSC319726 binds copper to induce oxidative stress and arrest glioblastoma-patient-derived cells at picomolar concentrations. Pharmacogenomic analysis suggested that NSC319726 and 65 other structural analogs exhibit lethality through metal binding. Although NSC319726 has been reported to function as a zinc ionophore, we report here that this compound binds to copper to arrest cell growth. We generated and validated pharmacogenomic predictions: copper toxicity was substantially inhibited by hypoxia, through an hypoxia-inducible-factor-1α-dependent pathway; copper-bound NSC319726 induced the generation of reactive oxygen species and depletion of deoxyribosyl purines, resulting in cell-cycle arrest. These results suggest that metal-induced DNA damage may be a consequence of exposure to some xenobiotics, therapeutic agents, as well as other causes of copper dysregulati...
Nature chemical biology, 2014
We previously discovered a small-molecule inducer of cell death, named 1541, that noncovalently s... more We previously discovered a small-molecule inducer of cell death, named 1541, that noncovalently self-assembles into chemical fibrils ('chemi-fibrils') and activates procaspase-3 in vitro. We report here that 1541-induced cell death is caused by the fibrillar rather than the soluble form of the drug. A short hairpin RNA screen reveals that knockdown of genes involved in endocytosis, vesicle trafficking and lysosomal acidification causes partial 1541 resistance. We confirm the role of these pathways using pharmacological inhibitors. Microscopy shows that the fluorescent chemi-fibrils accumulate in punctae inside cells that partially colocalize with lysosomes. Notably, the chemi-fibrils bind and induce liposome leakage in vitro, suggesting they may do the same in cells. The chemi-fibrils induce extensive proteolysis including caspase substrates, yet modulatory profiling reveals that chemi-fibrils form a distinct class from existing inducers of cell death. The chemi-fibrils shar...
Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recogni... more Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.
Precision medicine in oncology requires not only identification of cancer-associated mutations bu... more Precision medicine in oncology requires not only identification of cancer-associated mutations but also effective drugs for each cancer genotype, which is still a largely unsolved problem. One approach for the latter challenge has been large-scale testing of small molecules in genetically characterized cell lines. We hypothesized that compounds with high cell-line-selective lethality exhibited consistent results across such pharmacogenomic studies. We analyzed the compound sensitivity data of 6,259 lethal compounds from the NCI-60 project. A total of 2,565 cell-line-selective lethal compounds were identified and grouped into 18 clusters based on their median growth inhibitory GI50 profiles across the 60 cell lines, which were shown to represent distinct mechanisms of action. Further transcriptome analysis revealed a biomarker, NADPH abundance, for predicting sensitivity to ferroptosis-inducing compounds, which we experimentally validated. In summary, incorporating cell-line-selectivity filters improves the predictive power of pharmacogenomic analyses and enables discovery of biomarkers that predict the sensitivity of cells to specific cell death inducers.
Cell, 2014
Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We soug... more Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosisinducing 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.
ACS Medicinal Chemistry Letters, 2012
We analyzed more than 1 million small molecules with the goal of finding simple synthetic compoun... more We analyzed more than 1 million small molecules with the goal of finding simple synthetic compounds that potently inhibit cancer cell growth. We identified three such compounds with unknown mechanisms of action. Subsequent studies revealed that all three of these small molecules target microtubules. These three scaffolds can serve as templates for developing new microtubule-targeted agents, overcoming the limits of existing microtubule-inhibiting drugs derived from complex natural products.
Journal of the American Chemical Society, 2014
Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, 18 nonapoptotic cell ... more Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, 18 nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of 19 Huntington's disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; 20 Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species 21 formation or lysosomal membrane permeability. We developed a mechanistic model to 22 explain the activity of Fer-1, which guided the development of ferrostatins with improved 23 properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid 24 peroxidation mediates diverse disease phenotypes.
Cancer discovery, 2011
E3 ubiquitin ligases are of interest as drug targets for their ability to regulate protein stabil... more E3 ubiquitin ligases are of interest as drug targets for their ability to regulate protein stability and function. The oncogene Mdm2 is an attractive E3 ligase to target, as it is the key negative regulator of the tumor suppressor p53, which controls the transcription of genes involved in cell fate. Overexpression of Mdm2 facilitates tumorigenesis by inactivating p53, and through p53-independent oncogenic effects. We developed a high-throughput cellular Mdm2 auto-ubiquitination assay, which we used to discover a class of small-molecule Mdm2 ligase activity inhibitors. These compounds inhibit Mdm2 and p53 ubiquitination in cells, reduce viability of cells with wild-type p53, and synergize with DNA-damaging agents to cause cell death. We determined that these compounds effectively inhibit the E3 ligase activity of the Mdm2-MdmX hetero-complex. This mechanism may be exploitable to create a new class of anti-tumor agents.
A series of Pictet-Spengler condensation derivatives (tetrahydro-b-carbolines) was designed, synt... more A series of Pictet-Spengler condensation derivatives (tetrahydro-b-carbolines) was designed, synthesized and evaluated for lethality against a panel of seven cancer cell lines. Seven compounds (2a, 13, 20, 21, 27, 29 and 34) showed lethality in at least five cell lines. Among these, compound 27 showed a unique selectivity towards oncogenic-RAS expressing BJ-TERT/LT/ST/RAS V12 tumor cells, compared to non-transformed BJ-TERT cells. Further investigation revealed that 27 induces cell death without activation of caspases. This represents a useful new probe of non-apoptotic cell death and oncogenic-RAS synthetic lethality.
Cell death is a complex process that plays a vital role in development, homeostasis, and disease.... more Cell death is a complex process that plays a vital role in development, homeostasis, and disease. Our understanding of and ability to control cell death is impeded by an incomplete characterization of the full range of cell death processes that occur in mammalian systems, especially in response to exogenous perturbations. We present here a general approach to address this problem, which we call modulatory profiling. Modulatory profiles are composed of the changes in potency and efficacy of lethal compounds produced by a second cell death-modulating agent in human cell lines. We show that compounds with the same characterized mechanism of action have similar modulatory profiles. Furthermore, clustering of modulatory profiles revealed relationships not evident when clustering lethal compounds based on gene expression profiles alone. Finally, modulatory profiling of compounds correctly predicted three previously uncharacterized compounds to be microtubule-destabilizing agents, classified numerous compounds that act nonspecifically, and identified compounds that cause cell death through a mechanism that is morphologically and biochemically distinct from previously established ones. apoptosis | chemical biology | small molecules C ell death has historically been viewed as a binary phenomenon. Cells were described to die in one of two ways-through a controlled and ordered process (apoptosis) or an unregulated and chaotic process (necrosis) (1, 2). Not only were these often considered the only two possible mechanisms, but they were also frequently viewed as morphologically and biochemically uniform (3, 4). A great deal of research in recent decades has not only shown the complexity and heterogeneity of apoptotic and necrotic signaling, but also that cells can die in physiological and nonphysiological contexts through processes morphologically and biochemically distinct from both apoptosis and necrosis.