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.

RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels.

Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.

Functional model of metabolite gating by human voltage-dependent anion channel 2.

Voltage-dependent anion channels (VDACs) are critical regulators of outer mitochondrial membrane permeability in eukaryotic cells. VDACs have also been postulated to regulate cell death mechanisms. Erastin, a small molecule quinazolinone that is selectively lethal to tumor cells expressing mutant RAS, has previously been reported as a ligand for hVDAC2. While significant efforts have been made to elucidate the structure and function of hVDAC1, structural and functional characterization of hVDAC2 remains lacking. Here, we present an in vitro system that provides a platform for both functional and structural investigation of hVDAC2 and its small molecule modulator, erastin. Using this system, we found that erastin increases permeability of VDAC2 liposomes to NADH in a manner that requires the amino-terminal region of VDAC2. Furthermore, we confirmed that this VDAC2-lipsome sample is folded using solid-state NMR.

Development of Three Orthogonal Assays Suitable for the Identification and Qualification of PIKfyve Inhibitors.

FYVE-type zinc finger-containing phosphoinositide kinase (PIKfyve) catalyzes the formation of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol 3-phosphate (PI(3)P). PIKfyve has been implicated in multiple cellular processes, and its role in the regulation of toll-like receptor (TLR) pathways and the production of proinflammatory cytokines has sparked interest in developing small-molecule PIKfyve inhibitors as potential therapeutics to treat autoimmune and inflammatory diseases. We developed three orthogonal assays to identify and qualify small-molecule inhibitors of PIKfyve: (1) a purified component microfluidic enzyme assay that measures the conversion of fluorescently labeled PI(3)P to PI(3,5)P2 by purified recombinant full-length human 6His-PIKfyve (rPIKfyve); (2) an intracellular protein stabilization assay using the kinase domain of PIKfyve expressed in HEK293 cells; and (3) a cell-based functional assay that measures the production of interleukin (IL)-12p70 in human peripheral blood mononuclear cells stimulated with TLR agonists lipopolysaccharide and R848. We determined apparent Km values for both ATP and labeled PI(3)P in the rPIKfyve enzyme assay and evaluated the enzyme's ability to use phosphatidylinositol as a substrate. We also tested four reference compounds in the three assays and showed that together these assays provide a platform that is suitable to select promising inhibitors having appropriate functional activity and confirmed cellular target engagement to advance into preclinical models of inflammation.

SEC-TID: A Label-Free Method for Small-Molecule Target Identification.

Bioactive small molecules are an invaluable source of therapeutics and chemical probes for exploring biological pathways. Yet, significant hurdles in drug discovery often come from lacking a comprehensive view of the target(s) for both early tool molecules and even late-stage drugs. To address this challenge, a method is provided that allows for assessing the interactions of small molecules with thousands of targets without any need to modify the small molecule of interest or attach any component to a surface. We describe size-exclusion chromatography for target identification (SEC-TID), a method for accurately and reproducibly detecting ligand-macromolecular interactions for small molecules targeting nucleic acid and several protein classes. We report the use of SEC-TID, with a library consisting of approximately 1000 purified proteins derived from the protein databank (PDB), to identify the efficacy targets tankyrase 1 and 2 for the Wnt inhibitor XAV939. In addition, we report novel interactions for the tumor-vascular disrupting agent vadimezan/ASA404 (interacting with farnesyl pyrophosphate synthase) and the diuretic mefruside (interacting with carbonic anhydrase XIII). We believe this method can dramatically enhance our understanding of the mechanism of action and potential liabilities for small molecules in drug discovery pipelines through comprehensive profiling of candidate druggable targets.

Small Molecule that Reverses Dexamethasone Resistance in T-cell Acute Lymphoblastic Leukemia (T-ALL).

Glucocorticoids are one of the most utilized and effective therapies in treating T-cell acute lymphoblastic leukemia. However, patients often develop resistance to glucocorticoids, rendering these therapies ineffective. We screened 9517 compounds, selected for their lead-like properties, chosen from among 3 372 615 compounds, against a dexamethasone-resistant T-ALL cell line to identify small molecules that reverse glucocorticoid resistance. We synthesized analogues of the most effective compound, termed J9, from the screen in order to define the scaffold's structure-activity relationship. Active compounds restored sensitivity to glucocorticoids through upregulation of the glucocorticoid receptor. This compound and mechanism may provide a strategy for overcoming glucocorticoid resistance in patients with T-ALL.

Small molecule screen reveals regulation of survival motor neuron protein abundance by Ras proteins.

Small molecule modulators of protein activity have proven invaluable in the study of protein function and regulation. While inhibitors of protein activity are relatively common, small molecules that can increase protein abundance are rare. Small molecule protein upregulators with targeted activities would be of value in the study of the mechanisms underlying loss-of-function diseases. We developed a high-throughput screening approach to identify small molecule upregulators of the Survival of Motor Neuron protein (SMN), whose decreased levels cause the neurodegenerative disease spinal muscular atrophy (SMA). We screened 69,189 compounds for SMN upregulators and performed mechanistic studies on the most active compound, a bromobenzophenone analogue designated cuspin-1. Mechanistic studies of cuspin-1 revealed that increasing Ras signaling upregulates SMN protein abundance via an increase in translation rate. These findings suggest that controlled modulation of the Ras signaling pathway may benefit patients with SMA.

Mechanism of epidermal growth factor regulation of Vav2, a guanine nucleotide exchange factor for Rac.

Vav2 is a member of the Vav family that serves as a guanine nucleotide exchange factor for the Rho family of Ras-related GTPases. Unlike Vav1, whose expression is restricted to cells of hematopoietic origin, Vav2 is broadly expressed. Recently, Vav2 has been identified as a substrate for the epidermal growth factor (EGF) receptor; however, the mechanism by which Vav2 is activated in EGF-treated cells is unclear. By the means of an in vitro protein kinase assay, we show here that purified and activated EGF receptor phosphorylates Vav2 exclusively on its N-terminal domain. Furthermore, EGF receptor phosphorylates Vav2 on all three possible phosphorylation sites, Tyr-142, Tyr-159, and Tyr-172. In intact cells we also show that Vav2 associates with the activated EGF receptor in an Src homology 2 domain-dependent manner, with Vav2 Src homology 2 domain binding preferentially to autophosphorylation sites Tyr-992 and Tyr-1148 of the EGF receptor. Treatment of cells with EGF results in stimulation of exchange activity of Vav2 as measured on Rac; however, the intensity of the exchange activity does not show any correlation with the level of Vav2 tyrosine phosphorylation. Introducing a point mutation into the Vav2 pleckstrin homology domain or treatment of cells with the phosphatidylinositol 3-kinase inhibitor LY294002 prior to EGF stimulation inhibits Vav2 exchange activity. Although phosphorylation mutants of Vav2 can readily induce actin rearrangement in COS7 cells, pleckstrin homology domain mutant does not stimulate membrane ruffling. These results suggest that EGF regulates Vav2 activity basically through phosphatidylinositol 3-kinase activation, whereas tyrosine phosphorylation of Vav2 may rather be necessary for mediating protein-protein interactions.