RESUMO
Dysregulated oxidative stress plays a major role in cancer pathogenesis and some types of cancer cells are particularly vulnerable to inhibition of their cellular antioxidant capacity. Glutamate-cysteine ligase (GCL) is the first and rate-limiting step in the synthesis of the major cellular antioxidant glutathione (GSH). Developing a GCL inhibitor may be an attractive therapeutic strategy for certain cancer types that are particularly sensitive to oxidative stress. In this study, we reveal a cysteine-reactive ligand, EN25, that covalently targets an allosteric cysteine C114 on GCLM, the modifier subunit of GCL, and leads to inhibition of GCL activity. This interaction also leads to reduced cellular GSH levels and impaired cell viability in ARID1A-deficient cancer cells, which are particularly vulnerable to glutathione depletion, but not in ARID1A-positive cancer cells. Our studies uncover a novel potential ligandable site within GCLM that can be targeted to inhibit GSH synthesis in vulnerable cancer cell types.
Assuntos
Antioxidantes , Glutamato-Cisteína Ligase , Glutamato-Cisteína Ligase/metabolismo , Cisteína/metabolismo , Inibidores Enzimáticos , Glutationa/metabolismoRESUMO
ATP-competitive inhibitors that demonstrate exquisite selectivity for specific members of the human kinome have been developed. Despite this success, the identification of highly selective inhibitors is still very challenging, and it is often not possible to rationally engineer selectivity between the ATP-binding sites of kinases, especially among closely related family members. Src-family kinases (SFKs) are a highly homologous family of eight multidomain, nonreceptor tyrosine kinases that play general and specialized roles in numerous cellular processes. The high sequence and functional similarities between SFK members make it hard to rationalize how selectivity can be gained with inhibitors that target the ATP-binding site. Here, we describe the development of a series of inhibitors that are highly selective for the ATP-binding sites of the SFKs Lyn and Hck over other SFKs. By biochemically characterizing how these selective ATP-competitive inhibitors allosterically influence the global conformation of SFKs, we demonstrate that they most likely interact with a binding pocket created by the movement of the conformationally flexible helix αC in the ATP-binding site. With a series of sequence swap experiments, we show that sensitivity to this class of selective inhibitors is due to the identity of residues that control the conformational flexibility of helix αC rather than any specific ATP-binding site interactions. Thus, the ATP-binding sites of highly homologous kinases can be discriminated by targeting heterogeneity within conformationally flexible regions.
Assuntos
Trifosfato de Adenosina/metabolismo , Quinases da Família src/metabolismo , Sítios de Ligação , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/químicaRESUMO
The c-Jun N-terminal kinases (JNKs) play a wide variety of roles in cellular signaling processes, dictating important, and even divergent, cellular fates. These essential kinases possess docking surfaces distal to their active sites that interact with diverse binding partners, including upstream activators, downstream substrates, and protein scaffolds. Prior studies have suggested that the interactions of certain protein-binding partners with one such JNK docking surface, termed the D-recruitment site (DRS), can allosterically influence the conformational state of the ATP-binding pocket of JNKs. To further explore the allosteric relationship between the ATP-binding pockets and DRSs of JNKs, we investigated how the interactions of the scaffolding protein JIP1, as well as the upstream activators MKK4 and MKK7, are allosterically influenced by the ATP-binding site occupancy of the JNKs. We show that the affinity of the JNKs for JIP1 can be divergently modulated with ATP-competitive inhibitors, with a >50-fold difference in dissociation constant observed between the lowest- and highest-affinity JNK1-inhibitor complexes. Furthermore, we found that we could promote or attenuate phosphorylation of JNK1's activation loop by MKK4 and MKK7, by varying the ATP-binding site occupancy. Given that JIP1, MKK4, and MKK7 all interact with JNK DRSs, these results demonstrate that there is functional allostery between the ATP-binding sites and DRSs of these kinases. Furthermore, our studies suggest that ATP-competitive inhibitors can allosterically influence the intracellular binding partners of the JNKs.