Chemoproteomic Profiling of Covalent XPO1 Inhibitors to Assess Target Engagement and Selectivity.

Selinexor, a covalent XPO1 inhibitor, is approved in the USA in combination with dexamethasone for penta-refractory multiple myeloma. Additional XPO1 covalent inhibitors are currently in clinical trials for multiple diseases including hematologic malignancies, solid tumor malignancies, glioblastoma multiforme (GBM), and amyotrophic lateral sclerosis (ALS). It is important to measure the target engagement and selectivity of covalent inhibitors to understand the degree of engagement needed for efficacy, while avoiding both mechanism-based and off-target toxicity. Herein, we report clickable probes based on the XPO1 inhibitors selinexor and eltanexor for the labeling of XPO1 in live cells to assess target engagement and selectivity. We used mass spectrometry-based chemoproteomic workflows to profile the proteome-wide selectivity of selinexor and eltanexor and show that they are highly selective for XPO1. Thermal profiling analysis of selinexor further offers an orthogonal approach to measure XPO1 engagement in live cells. We believe these probes and assays will serve as useful tools to further interrogate the biology of XPO1 and its inhibition in cellular and in vivo systems.

NonA and CPX Link the Circadian Clockwork to Locomotor Activity in Drosophila.

Drosophila NonA and its mammalian ortholog NONO are members of the Drosophila behavior and human splicing (DBHS) family. NONO also has a strong circadian connection: it associates with the circadian repressor protein PERIOD (PER) and contributes to circadian timekeeping. Here, we investigate NonA, which is required for proper levels of evening locomotor activity as well as a normal free-running period in Drosophila. NonA is associated with the positive transcription factor CLOCK/CYCLE (CLK/CYC), interacts directly with complexin (cpx) pre-mRNA, and upregulates gene expression, including the gene cpx. Downregulation of cpx expression in circadian neurons phenocopies NonA downregulation, whereas cpx overexpression rescues the nonA RNAi phenotypes, indicating that cpx is an important NonA target gene. As the cpx protein contributes to proper neurotransmitter and neuropeptide release in response to calcium, these results and others indicate that this control is important for the normal circadian regulation of locomotor activity.

Chemoproteomic Strategy to Quantitatively Monitor Transnitrosation Uncovers Functionally Relevant S-Nitrosation Sites on Cathepsin D and HADH2.

S-Nitrosoglutathione (GSNO) is an endogenous transnitrosation donor involved in S-nitrosation of a variety of cellular proteins, thereby regulating diverse protein functions. Quantitative proteomic methods are necessary to establish which cysteine residues are most sensitive to GSNO-mediated transnitrosation. Here, a competitive cysteine-reactivity profiling strategy was implemented to quantitatively measure the sensitivity of >600 cysteine residues to transnitrosation by GSNO. This platform identified a subset of cysteine residues with a high propensity for GSNO-mediated transnitrosation. Functional characterization of previously unannotated S-nitrosation sites revealed that S-nitrosation of a cysteine residue distal to the 3-hydroxyacyl-CoA dehydrogenase type 2 (HADH2) active site impaired catalytic activity. Similarly, S-nitrosation of a non-catalytic cysteine residue in the lysosomal aspartyl protease cathepsin D (CTSD) inhibited proteolytic activation. Together, these studies revealed two previously uncharacterized cysteine residues that regulate protein function, and established a chemical-proteomic platform with capabilities to determine substrate specificity of other cellular transnitrosation agents.

Chemical-proteomic strategies to investigate cysteine posttranslational modifications.

The unique combination of nucleophilicity and redox-sensitivity that is characteristic of cysteine residues results in a variety of posttranslational modifications (PTMs), including oxidation, nitrosation, glutathionylation, prenylation, palmitoylation and Michael adducts with lipid-derived electrophiles (LDEs). These PTMs regulate the activity of diverse protein families by modulating the reactivity of cysteine nucleophiles within active sites of enzymes, and governing protein localization between soluble and membrane-bound forms. Many of these modifications are highly labile, sensitive to small changes in the environment, and dynamic, rendering it difficult to detect these modified species within a complex proteome. Several chemical-proteomic platforms have evolved to study these modifications and enable a better understanding of the diversity of proteins that are regulated by cysteine PTMs. These platforms include: (1) chemical probes to selectively tag PTM-modified cysteines; (2) differential labeling platforms that selectively reveal and tag PTM-modified cysteines; (3) lipid, isoprene and LDE derivatives containing bioorthogonal handles; and (4) cysteine-reactivity profiling to identify PTM-induced decreases in cysteine nucleophilicity. Here, we will provide an overview of these existing chemical-proteomic strategies and their effectiveness at identifying PTM-modified cysteine residues within native biological systems.