Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Caspase-9 swings both ways in the apoptosome.

For nearly 2 decades, investigators have debated whether cysteinyl-aspartate-specific protease 9 (caspase-9) is activated within the apoptotic protease-activating factor 1 (Apaf-1) apoptosome through proximity-induced homodimerization or through formation of a holoenzyme. Recently, we have demonstrated that caspase-9 forms (and likely transitions between) both caspase-9 homo- and Apaf-1:caspase-9 heterodimers, each of which plays unique roles in the recruitment and activation of caspase-9.

The Apaf-1 apoptosome induces formation of caspase-9 homo- and heterodimers with distinct activities.

According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.

Regulation of the intrinsic apoptosis pathway by reactive oxygen species.

SIGNIFICANCE: The intrinsic apoptosis pathway is conserved from worms to humans and plays a critical role in the normal development and homeostatic control of adult tissues. As a result, numerous diseases from cancer to neurodegeneration are associated with either too little or too much apoptosis. RECENT ADVANCES: B cell lymphoma-2 (BCL-2) family members regulate cell death, primarily via their effects on mitochondria. In stressed cells, proapoptotic BCL-2 family members promote mitochondrial outer membrane permeabilization (MOMP) and cytochrome c (cyt c) release into the cytoplasm, where it stimulates formation of the "apoptosome." This large, multimeric complex is composed of the adapter protein, apoptotic protease-activating factor-1, and the cysteine protease, caspase-9. Recent studies suggest that proteins involved in the processes leading up to (and including) formation of the apoptosome are subject to various forms of post-translational modification, including proteolysis, phosphorylation, and in some cases, direct oxidative modification. CRITICAL ISSUES: Despite intense investigation of the intrinsic pathway, significant questions remain regarding how cyt c is released from mitochondria, how the apoptosome is formed and regulated, and how caspase-9 is activated within the complex. FUTURE DIRECTIONS: Further studies on the biochemistry of MOMP and apoptosome formation are needed to understand the mechanisms that underpin these critical processes, and novel animal models will be necessary in the future to ascertain the importance of the many posttranslational modifications reported for BCL-2 family members and components of the apoptosome.

Inactivation of ataxia telangiectasia mutated gene can increase intracellular reactive oxygen species levels and alter radiation-induced cell death pathways in human glioma cells.

PURPOSE: To investigate the effects of ataxia telangiectasia mutated (ATM)-regulated reactive oxygen species (ROS) and cell death pathways on the response of U87MG glioma cells to ionising radiation (IR) and oxidative stress. MATERIAL AND METHODS: ATM expression was blocked in U87MG glioma cells using a small interfering RNA (siRNA) technique. Cell survival, sub-lethal damage (SLD), and potential lethal damage (PLD) repair following IR were assessed by clonogenic assay while changes in intracellular ROS, the apoptosis, and autophagy were followed by flow cytometry and Western blotting. RESULTS: Blocking ATM expression in U87MG cells increased intracellular ROS levels and sensitivity to the cytotoxic effects of IR and oxygen stress; effects that could be partly counteracted by the antioxidant N-acetylcysteine (NAC). Knock down of ATM rendered cells unable to repair sub-lethal or potentially lethal damage and DNA double strand breaks (DSB) after IR exposure; something that NAC could not counteract. ATM did control the pathways a cell used to die following IR and this did seem to be ROS-dependent. CONCLUSION: ATM is involved in redox control but ROS elevations following ATM knock down seem more involved in the decision as to what cell death pathway is utilised after IR than DSB repair and radiosensitivity.

DARK apoptosome secrets come to light.

In this issue of Structure, Yuan et al. (2011) utilize biochemical approaches to reconstitute an active Drosophila apoptosome, as well as cryo-electron microscopy to generate an improved model for this conserved caspase-activating complex.