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 1 . 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 * These authors contributed equally to this work.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions N.Y. designed and performed the RNAi and VDAC overexpression, quantitative PCR, erastin analogue viability and chemical characterization experiments. E.Z. performed two-dimensional western analysis, PARP-1 and pro-caspase-3 cleavage, and cytochrome c release experiments. E.Z. and N.Y. performed transmission electron microscopy experiments. A.J.B., D.J.F. and N.Y. performed the NADH oxidation and direct binding experiments. W.S.Y. characterized sensitivity to erastin in the BJderived cell series. A.J.W. performed the MEK1/2 inhibitor experiment. I.S. and A.J.B. synthesized erastin analogues. R.S. and S.L.L. provided BRAF shRNAs, analysis of BRAF knockdown and the phospho-ERK western analysis. J.M.P., J.J.B. and S.S. were responsible for setting up the technology platform to pull down proteins binding to small molecule compounds. M.v.R. and J.M.P. performed the pull-down experiments. J.J.B., J.M.P. and S.S. designed, reviewed and supervised the pull-down experiments, and contributed to the analysis of the data. B.R.S. conceived of and supervised the project, designed and analysed experiments, and performed the anti-oxidant studies. B.R.S. and N.Y. prepared the manuscript. (Fig. 1a , Supplementary Fig. 1 and ref. 3 ). This cell death was not dependent on the rate of cell division, nor was it idiosyncratic to these cells ( Fig. 1a and Supplementary Fig. 2), because cell lines engineered in a similar way responded similarly. Author InformationWe found th...

Chemical genetics is an emerging approach for studying biological systems using chemical tools. This strategy aims to reveal the macromolecules responsible for regulating biological systems; thus, the approach shares much in common with genetics. In both strategies, one must (a) develop an assay that reports on a biological process of interest, (b) perturb this process systematically (with mutations or small molecules), and (c) determine the target of each perturbation to reveal macromolecules (i.e., proteins and genes) regulating the process of interest. In this review, we discuss advances and challenges in this field that have emerged over the past four years. Several technologies have converged, raising the hope that it may be possible to systematically apply chemical probes to biological processes.

Synthetic methodology was developed to construct amino acid-[2]rotaxanes that have phenylalanine and 3,5-di-tert-butylbenzene as blocking groups and dibenzo-24-crown-8, derivatized with either N-acetylargininyl or a carboxylic group, as the ring. A relative measure of the intramolecular interaction energies between the functional groups in DMSO/water mixtures is obtained by comparing their pK(a) values. Rotaxane structures were investigated through 2D NMR analysis and molecular dynamics simulations. Association constants for complexes of amino acids and rotaxanes in various protonation states were determined in a variety of solvent systems by (1)H NMR analysis. The unique intracomponent interactions that exist in the rotaxanes and their ability to act as artificial receptors are discussed.

A host-[2]rotaxane was constructed by converting a diaminophenylcalix[4]arene into a [2]rotaxane using the DCC-rotaxane method (Zehnder, D.; Smithrud, D. B. Org. Lett. 2001, 16, 2485-2486). N-Ac-Arg groups were attached to the dibenzo-24-crown-8 ring of the rotaxane to provide a convergent functional group. To demonstrate the advantage provided by the rotaxane architecture for recognition of guests that contain a variety of functional groups, association constants (K(A)) for N-Ac-Trp, indole, N-Ac-Gly, fluorescein, 1-(dimethylamino)-5-naphthalenesulfonate, and pyrene bound to the [2]rotaxane were determined by performing (1)H NMR and fluorescence spectroscopic experiments. The host-[2]rotaxane had the highest affinity for fluorescein with a K(A) = 4.6 x 10(6) M(-)(1) in a 98/2 buffer (1 mM phosphate, pH 7)/DMSO solution. A comparison of K(A) values demonstrates that both the aromatic pocket and ring of the host-[2]rotaxane contribute binding free energy for complexation. Association constants were also derived for the same guests bound to the diaminophenylcalix[4]arene and to a diphenylcalix[4]arene that contained arginine residues displayed in a nonconvergent fashion. The host-[2]rotaxane provides higher affinity and specificity for most guests than the host with divergent N-Ac-Arg groups of the one that only has an aromatic pocket. For example, the K(A) for the complex of the host-[2]rotaxane and fluorescein in the DMSO/water mixture is more than 2 orders of magnitude greater than association constants derived for the other hosts.