Identification of Tunable, Environmentally Responsive Fluorogenic Dyes by High-Throughput Screening.

Small molecule dyes remain essential biological tools, yet only a handful of environmentally responsive fluorogenic small molecules are available for routine characterization of protein state. Here, we report the development and execution of a high throughput screen to identify compounds that increase in fluorescence in response to binding of lipophilic sites of proteins. This effort yielded two small molecules that potently indicate the presence of a range of common proteins and outperform common dyes in differential scanning fluorimetry experiments. Structure activity relationship studies revealed that these two scaffolds can be tuned both for their quantum yields and emission wavelengths. This work affords a straightforward framework for the discovery of new fluorophores and adds two fluorogenic probes to the toolbox for studying protein state.

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.

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.

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.

Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2.

Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may therefore display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP dependent cellular growth.

Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2.

Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may, therefore, display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP-driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP-dependent cellular growth.

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.