Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe.

Covalent inhibitors of enzymes are increasingly appreciated as pharmaceutical seeds, yet discovering non-cysteine-targeting inhibitors remains challenging. Herein, we report an intriguing experience during our activity-based proteomic screening of 1601 reactive small molecules, in which we monitored the ability of library molecules to compete with a cysteine-reactive iodoacetamide probe. One epoxide molecule, F8, exhibited unexpected enhancement of the probe reactivity for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-limiting glycolysis enzyme. In-depth mechanistic analysis suggests that F8 forms a covalent adduct with an aspartic acid in the active site to displace NAD+, a cofactor of the enzyme, with concomitant enhancement of the probe reaction with the catalytic cysteine. The mechanistic underpinning permitted the identification of an optimized aspartate-reactive GAPDH inhibitor. Our findings exemplify that activity-based proteomic screening with a cysteine-reactive probe can be used for discovering covalent inhibitors that react with non-cysteine residues.

Physical and Functional Analysis of the Putative Rpn13 Inhibitor RA190.

Rpn13 is one of several ubiquitin receptors in the 26S proteasome. Cys88 of Rpn13 has been proposed to be the principal target of RA190, an electrophilic small molecule with interesting anti-cancer activities. Here, we examine the claim that RA190 mediates its cytotoxic effects through engagement with Rpn13. We find no evidence that this is the case. In vitro, RA190 is has no measurable effect on any of the known interactions of Rpn13. In cellulo, we see no physical engagement of Rpn13 by RA190, either on C88 or any other residue. However, chemical proteomics experiments in two different cell lines reveal that dozens of other proteins are heavily engaged by RA190. Finally, increasing or reducing the level of Rpn13 in HeLa and melanoma cells had no effect on the sensitivity of HeLa or melanoma cells to RA190. We conclude that Rpn13 is not the physiologically relevant target of RA190.

Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4-MyD88 Complex.

Dimethyl fumarate (DMF) is a prescribed treatment for multiple sclerosis and has also been used to treat psoriasis. The electrophilicity of DMF suggests that its immunosuppressive activity is related to the covalent modification of cysteine residues in the human proteome. Nonetheless, our understanding of the proteins modified by DMF in human immune cells and the functional consequences of these reactions remains incomplete. In this study, we report that DMF inhibits human plasmacytoid dendritic cell function through a mechanism of action that is independent of the major electrophile sensor NRF2. Using chemical proteomics, we instead identify cysteine 13 of the innate immune kinase IRAK4 as a principal cellular target of DMF. We show that DMF blocks IRAK4-MyD88 interactions and IRAK4-mediated cytokine production in a cysteine 13-dependent manner. Our studies thus identify a proteomic hotspot for DMF action that constitutes a druggable protein-protein interface crucial for initiating innate immune responses.

Small-molecule screening yields a compound that inhibits the cancer-associated transcription factor Hes1 via the PHB2 chaperone.

The transcription factor Hes family basic helix-loop-helix transcription factor 1 (Hes1) is a downstream effector of Notch signaling and plays a crucial role in orchestrating developmental processes during the embryonic stage. However, its aberrant signaling in adulthood is linked to the pathogenesis of cancer. In the present study, we report the discovery of small organic molecules (JI051 and JI130) that impair the ability of Hes1 to repress transcription. Hes1 interacts with the transcriptional corepressor transducing-like enhancer of split 1 (TLE1) via an interaction domain comprising two tryptophan residues, prompting us to search a chemical library of 1,800 small molecules enriched for indole-like π-electron-rich pharmacophores for a compound that blocks Hes1-mediated transcriptional repression. This screening identified a lead compound whose extensive chemical modification to improve potency yielded JI051, which inhibited HEK293 cell proliferation with an EC50 of 0.3 µm Unexpectedly, using immunomagnetic isolation and nanoscale LC-MS/MS, we found that JI051 does not bind TLE1 but instead interacts with prohibitin 2 (PHB2), a cancer-associated protein chaperone. We also found that JI051 stabilizes PHB2's interaction with Hes1 outside the nucleus, inducing G2/M cell-cycle arrest. Of note, JI051 dose-dependently reduced cell growth of the human pancreatic cancer cell line MIA PaCa-2, and JI130 treatment significantly reduced tumor volume in a murine pancreatic tumor xenograft model. These results suggest a previously unrecognized role for PHB2 in the regulation of Hes1 and may inform potential strategies for managing pancreatic cancer.

A Potent and Site-Selective Agonist of TRPA1.

TRPA1 is a member of the transient receptor potential (TRP) cation channel family that is expressed primarily on sensory neurons. This chemosensor is activated through covalent modification of multiple cysteine residues with a wide range of reactive compounds including allyl isothiocyanate (AITC), a spicy component of wasabi. The present study reports on potent and selective agonists of TRPA1, discovered through screening 1657 electrophilic molecules. In an effort to validate the mode of action of hit molecules, we noted a new TRPA1-selective agonist, JT010 (molecule 1), which opens the TRPA1 channel by covalently and site-selectively binding to Cys621 (EC50 = 0.65 nM). The results suggest that a single modification of Cys621 is sufficient to open the TRPA1 channel. The TRPA1-selective probe described herein might be useful for further mechanistic studies of TRPA1 activation.

Synthetic molecules that protect cells from anoikis and their use in cell transplantation.

One of the major problems encountered in cell transplantation is the low level of survival of transplanted cells due to detachment-induced apoptosis, called anoikis. The present study reports on the chemical synthesis and biological evaluation of water-soluble molecules that protect suspended cells from anoikis. The synthetic molecules bind to and induce clusters of integrins and heparan-sulfate-bound syndecans, two classes of receptors that are important for extracellular matrix-mediated cell survival. Molecular biological analysis indicates that such molecules prolong the survival of suspended NIH3T3 cells, at least in part, by promoting clustering of syndecan-4 and integrin ß1 on the cell surface, leading to the activation of small GTPase Rac-1 and Akt. In vivo experiments using animal disease models demonstrated the ability of the molecules to improve cell engraftment. The cluster-inducing molecules may provide a starting point for the design of new synthetic tools for cell-based therapy.