Covalent Targeting As a Common Mechanism for Inhibiting NLRP3 Inflammasome Assembly.

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines, including IL-1ß and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remain poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit the inflammasome assembly and activity. From this screen, we identify and profile inflammasome inhibition of 20 new covalent compounds across nine different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks the activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular cross-linking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, further support the continued development of electrophilic compounds that target reactive cysteine residues on NLRP3 to regulate its activation and activity.

A covalent inhibitor of the YAP-TEAD transcriptional complex identified by high-throughput screening.

Yes-associated protein (YAP), the master transcriptional effector downstream of the Hippo pathway, regulates essential cell growth and regenerative processes in animals. However, the activation of YAP observed in cancers drives cellular proliferation, metastasis, chemoresistance, and immune suppression, making it of key interest in developing precision therapeutics for oncology. As such, pharmacological inhibition of YAP by targeting its essential co-regulators, TEA domain transcription factors (TEADs) would likely promote tumor clearance in sensitive tumor types. From a fluorescence polarization-based high throughput screen of over 800 000 diverse small molecules, here we report the identification of a pyrazolopyrimidine-based scaffold that inhibits association of YAP and TEADs. Medicinal chemistry-based optimization identified mCMY020, a potent, covalent inhibitor of TEAD transcriptional activity that occupies a conserved, central palmitoylation site on TEADs.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.

Cross talk between metabolism and stress-responsive signaling is essential for maintaining cellular homeostasis. This cross talk is often achieved through covalent modification of proteins by endogenous, reactive metabolites that regulate key stress-responsive transcription factors like NRF2. Metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 repressor KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolism to NRF2 activation. Succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

Covalent targeting as a common mechanism for inhibiting NLRP3 inflammasome assembly.

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines including IL-1ß and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remains poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit inflammasome assembly and activity. From this screen we identify and profile inflammasome inhibition of 20 new covalent compounds across 9 different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular crosslinking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, suggests that NLRP3 serves as a cellular electrophile sensor important for coordinating inflammatory signaling in response to redox stress. Further, our results support the potential for covalent cysteine modification of NLRP3 for regulating inflammasome activation and activity.

S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling.

KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.

Crosstalk between metabolism and stress-responsive signaling is essential to maintaining cellular homeostasis. One way this crosstalk is achieved is through the covalent modification of proteins by endogenous, reactive metabolites that regulate the activity of key stress-responsive transcription factors such as NRF2. Several metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 regulatory protein KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolic pathways to NRF2 activation. We found that succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 transcriptional signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

Metabolically Activated Proteostasis Regulators Protect against Glutamate Toxicity by Activating NRF2.

The extracellular accumulation of glutamate is a pathologic hallmark of numerous neurodegenerative diseases including ischemic stroke and Alzheimer's disease. At high extracellular concentrations, glutamate causes neuronal damage by promoting oxidative stress, which can lead to cellular death. This has led to significant interest in developing pharmacologic approaches to mitigate the oxidative toxicity caused by high levels of glutamate. Here, we show that the small molecule proteostasis regulator AA147 protects against glutamate-induced cell death in a neuronal-derived cell culture model. While originally developed as an activator of the activating transcription factor 6 (ATF6) arm of the unfolded protein response, this AA147-dependent protection against glutamate toxicity is primarily mediated through activation of the NRF2-regulated oxidative stress response. We demonstrate that AA147 activates NRF2 selectively in neuronal-derived cells through a mechanism involving metabolic activation to a reactive electrophile and covalent modification of KEAP1─a mechanism analogous to that involved in the AA147-dependent activation of ATF6. These results define the potential for AA147 to protect against glutamate-induced oxidative toxicity and highlight the potential for metabolically activated proteostasis regulators like AA147 to activate both protective ATF6 and NRF2 stress-responsive signaling pathways to mitigate oxidative damage associated with diverse neurologic diseases.

YAP-dependent proliferation by a small molecule targeting annexin A2.

The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.