Small molecule probes for cellular death machines

Abstract

The past decade has witnessed a significant expansion of our understanding about the regulated cell death mechanisms beyond apoptosis. The application of chemical biological approaches had played a major role in driving these exciting discoveries. The discovery and use of small molecule probes in cell death research has not only revealed significant insights into the regulatory mechanism of cell death but also provided new drug targets and lead drug candidates for developing therapeutics of human diseases with huge unmet need. Here, we provide an overview of small molecule modulators for necroptosis and ferroptosis, two non-apoptotic cell death mechanisms, and discuss the molecular pathways and relevant pathophysiological mechanisms revealed by the judicial applications of such small molecule probes. We suggest that the development and applications of small molecule probes for non-apoptotic cell death mechanisms provide an outstanding example showcasing the power of chemical biology in exploring novel biological mechanisms.

Figure 1

The necroptosis pathway and small molecule inhibitors of necroptosis. The binding of TNFα to its cognate receptor TNFR1 triggers the assembly of a membrane associated complex composed of TRADD, RIPK1, TRAF2, cIAPs and CYLD, which promotes the activation of RIPK1. Under apoptosis deficient conditions, activated RIPK1 binds to RIPK3 and stimulates its phosphorylation, which in turn phosphorylates a pseudo-kinase MLKL. Phosphorylated MLKL promotes its oligomerization and translocation from cytosol to cytoplasmic and internal membrane compartments to execute necroptosis. The Hsp90/Cdc37 complex promotes necroptosis by facilitating the folding and maturation of three core components of necroptotic machine, RIPK1, RIPK3 and MLKL. Nec-1 and its improved analog Nec-1s, the clinical candidate GSK2982772, a hybrid molecule PN10 consisting of pharmacophores of Nec-1s and ponatinib, and the type II RIPK1 inhibitor Cpd27 from GSK all inhibit the kinase activity of RIPK1, thereby preventing necroptosis. Additional necroptosis inhibitors include RIPK3 kinase inhibitors GSK′840, GSK′843 and GSK′872, the covalent inhibitor of MLKL NSA, the ATP competitive inhibitor of MLKL GW806742X, and Hsp90 inhibitors 17-AAG and Kongensin A.

Figure 2

The ferroptosis pathway and the inducers and inhibitors of ferroptosis. Ferroptosis is induced by iron-dependent accumulation of lethal lipid peroxidation products due to the disruption of the essential cellular antioxidative defense mechanism. Under normal conditions, the uptake of cystine through system x_c⁻ and the subsequent reduction of cystine to cysteine are critical for the synthesis of the major antioxidant GSH. GPx4, the major enzyme that detoxifies lipid ROS, converts GSH to oxidized glutathione and reduces lipid peroxides to their corresponding hydroxyl derivatives. On the other hand, ACLS4 (acyl-CoA synthetase long-chain family member 4) promotes the production of polyunsaturated fatty acids which may be converted to phospholipids sensitive to oxygenation by lipoxygenases (LOX12/15). The import and export of iron is mediated by TfR1 and Ferroportin respectively. Changes in iron transport can lead to iron overload that induces ferroptosis. Excessive iron in its labile form can catalyze the Fenton reaction to produce free radicals or act as cofactors for LOXs to promote ferroptosis through the Fenton reaction to generate lipid ROS. A series of ferroptosis inducers has been shown to interfere with the anti-oxidant network at different upstream events. Erastin, Glutamate, sorafenin and lanperisone inhibit cytstine uptake through system x_c⁻, BSO blocks the biosynthesis of GSH, and RSL3 inactivate GPx4. Ferroptosis can also be inhibited by a variety of ferroptosis inhibitors. CPO, DFO, Deferasirox and deferiprone inhibit ferroptosis by chelating irons. NDGA, Zileuton, AA861, BW A4C and PD 146176 inhibit LOX 12/15. ROSI, PIO and TRO, known PPARγ agonists, inhibit ACSL4 and alleviate ferroptosis independent of PPARγ. Ferrostatins, Liproxstatins, trolox, α-tocopherol, BHT, β-carotene and Ebs function as antioxidants to protect cells from ferroptosis.