Project description:Current methods for measuring amino-acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts, and software to boost data acquisition in real-time on the mass spectrometer. Our method, Streamlined Cysteine Activity-Based Protein Profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites in 18 min per compound. We applied it to identifying the proteome-wide targets of a covalent inhibitor of mutant KRASG12C and of ibrutinib, a covalent inhibitor of BTK. In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines , which includes >20,000 cysteines in >6,000 proteins per cell line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.
Project description:Chemoproteomics investigates small molecule-protein interactions and has made significant progress in recent years. Despite its vast potential, the proteome-wide profiling of reactive cysteine ligandability remains a formidable task to adapt for high throughput applications. This is primarily due to a lack of platforms capable of achieving the desired depth using low sample input in 96- or 384-well plates. Here we have revamped the cysteine profiling platform to address the challenge with an eye toward performing high-throughput library screening in plates. By incorporating several changes including i) an 18-plex TMT sample multiplexing strategy, ii) a magnetic beads-based one-pot workflow, iii) a 10X higher capacity streptavidin resin, and iv) optimized mass spectrometry analyses, a plate-based platform was developed that enables routine interrogation of either ~18,000 or ~24,000 reactive cysteines based on starting amounts of 10 or 20 µg, respectively. We applied the platform to screen a library of 192 electrophiles in the native HEK293T proteome, mapping the ligandablity of 38,450 reactive cysteines from 8,274 human proteins. The significantly improved depth revealed many previously unknown reactive cysteines and cysteine-ligand interactions and led to the identification of an azepane-containing acrylamide which has preferential binding to cysteines in EGF-like domains. We further applied the platform to characterize new cellular targets of well-studied compounds and covalent drugs in three different human cell lines. We found that ARS-1620, a KRASG12C inhibitor, also binds to cysteine 140 of an off-target adenosine kinase ADK, inhibiting its kinase activity. The platform represents a major step forward to high throughput evaluation of reactive cysteines on a proteome-wide scale.
Project description:Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is associated with enhanced expression during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. When added to cells, LN5P45 shows outstanding target engagement and proteome-wide selectivity. Importantly, we found that LN5P45 and the other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 via lysine 31. Thus, our findings provide novel insights for the design of future OTUB2-related therapeutics and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.
Project description:Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap, an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFkB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting,and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity
Project description:isoDTB-ABPP Cysteine Chemoproteomic Profiling was used to detect the targets of bigelovin in NLRP3 inflammasome activation. LPS and ATP induced bone marrow derived macrophages were adopted for the experiment in three biological replicates. Cell was treated with DMSO and bigelovin respectively. The cell lysate was incubated with IA-alkyne, followed by click reaction. The DMSO group was labeled by light isoDTB tags and the bigelovin group was labeled by heavy isoDTB tags.
Project description:First experiment: Cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM methionine + 0.1 mM cysteine (complete) or supplemented only with 0.1 mM methionine (cysteine-free). Cells were cultured in either medium for 42 h (Long + Cys; Long -Cys) or in cysteine-free medium for 36 h followed by 6 h in complete medium (Short +Cys) Second experiment: C3A/HepG2 cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM Met and 0.1 mM Cys (complete) or supplemented only with 0.1 mM Met (cysteine-devoid). Cells were cultured in complete medium for 42 h (Long +Cys) or in complete medium for 36 h followed by cysteine-devoid medium for 6 h (Short -Cys). Keywords: amino acid deprivation
Project description:First experiment: Cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM methionine + 0.1 mM cysteine (complete) or supplemented only with 0.1 mM methionine (cysteine-free). Cells were cultured in either medium for 42 h (Long + Cys; Long -Cys) or in cysteine-free medium for 36 h followed by 6 h in complete medium (Short +Cys); Second experiment: C3A/HepG2 cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM Met and 0.1 mM Cys (complete) or supplemented only with 0.1 mM Met (cysteine-devoid). Cells were cultured in complete medium for 42 h (Long +Cys) or in complete medium for 36 h followed by cysteine-devoid medium for 6 h (Short -Cys). Experiment Overall Design: First experiment: Three plates of cells were cultured under each condition. Cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM methionine + 0.1 mM cysteine (complete) or supplemented only with 0.1 mM methionine (cysteine-free). Cells were cultured in either medium for 42 h (Long + Cys; Long -Cys) or in cysteine-free medium for 36 h followed by 6 h in complete medium (Short +Cys). Experiment Overall Design: Second experiment: C3A/HepG2 cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM Met and 0.1 mM Cys (complete) or supplemented only with 0.1 mM Met (cysteine-devoid). Cells were cultured in complete medium for 42 h (Long +Cys) or in complete medium for 36 h followed by cysteine-devoid medium for 6 h (Short -Cys).