Covalent Targeting of Splicing in T Cells

Kevin A. Scott, Hiroyuki Kojima, Nathalie Ropek, Charles D. Warren, Tiffany L. Zhang, Simon J. Hogg, Caroline Webster, Xiaoyu Zhang, Jahan Rahman, Bruno Melillo, Benjamin F. Cravatt, Jiankun Lyu, Omar Abdel-Wahab, Ekaterina V Vinogradova

bioRxiv 2023.12.18.572199; 

doi: https://doi.org/10.1101/2023.12.18.572199

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2 inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

Defining the Cell Surface Cysteinome Using Two-Step Enrichment Proteomics

Tianyang Yan, Lisa M. Boatner, Liujuan Cui, Peter J. Tontonoz, and Keriann M. Backus

JACS Au 2023

DOI: 10.1021/jacsau.3c00707

The plasma membrane proteome is a rich resource of functionally important and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here, we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surface Cysteinome. Our “Cys-Surf” platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of low-density lipoprotein (LDL) particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome.

Electrophile Scanning Reveals Reactivity Hotspots for the Design of Covalent Peptide Binders

Nathalie M. Grob, Clint Remarcik, Simon L. Rössler, Jeffrey Y. K. Wong, John C. K. Wang, Jason Tao, Corey L. Smith, Andrei Loas, Stephen L. Buchwald, Dan L. Eaton, Magdalena Preciado López, and Bradley L. Pentelute

ACS Chemical Biology 2023

DOI: 10.1021/acschembio.3c00538

Protein–protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets. To improve binding interactions with these topologies and advance the development of PPI-focused therapeutics, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. We report a strategy termed electrophile scanning to identify reactivity hotspots in a known peptide ligand and demonstrate its application in a model PPI. Cysteine mutants of a known ligand are used to install protein-reactive modifiers via a palladium oxidative addition complex (Pd-OAC). Reactivity hotspots are revealed by cross-linking reactions with the target protein under physiological conditions. In a model PPI with the 9-mer peptide antigen VL9 and major histocompatibility complex (MHC) class I protein HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein–peptide conjugate within 4 h. The reactions are specific to the target protein in vitro and dependent on the peptide sequence. Moreover, the cross-linked peptide successfully inhibits molecular recognition of HLA-E by CD94–NKG2A possibly due to structural changes enacted at the PPI interface. The results illustrate the potential application of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.

Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics

Qianni Peng, Eranthie Weerapana 

Cell Chemical Biology, 2023

DOI: https://doi.org/10.1016/j.chembiol.2023.11.010

The nucleus controls cell growth and division through coordinated interactions between nuclear proteins and chromatin. Mutations that impair nuclear protein association with chromatin are implicated in numerous diseases. Covalent ligands are a promising strategy to pharmacologically target nuclear proteins, such as transcription factors, which lack ordered small-molecule binding pockets. To identify nuclear cysteines that are susceptible to covalent liganding, we couple proximity labeling (PL), using a histone H3.3-TurboID (His-TID) construct, with chemoproteomics. Using covalent scout fragments, KB02 and KB05, we identified ligandable cysteines on proteins involved in spindle assembly, DNA repair, and transcriptional regulation, such as Cys101 of histone acetyltransferase 1 (HAT1). Furthermore, we show that covalent fragments can affect the abundance, localization, and chromatin association of nuclear proteins. Notably, the Parkinson disease protein 7 (PARK7) showed increased nuclear localization and chromatin association upon KB02 modification at Cys106. Together, this platform provides insights into targeting nuclear cysteines with covalent ligands.

Discovery of YSR734: A Covalent HDAC Inhibitor with Cellular Activity in Acute Myeloid Leukemia and Duchenne Muscular Dystrophy

Yasir S. Raouf, Abootaleb Sedighi, Mulu Geletu, Geordon A. Frere, Rebecca G. Allan, Nabanita Nawar, Elvin D. de Araujo, and Patrick T. Gunning

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.3c01236

Histone deacetylases (HDACs) have emerged as powerful epigenetic modifiers of histone/non-histone proteins via catalyzing the deacetylation of ε-N-acetyl lysines. The dysregulated activity of these Zn2+-dependent hydrolases has been broadly implicated in disease, notably cancer. Clinically, the recurring dose-limiting toxicities of first-generation HDACi sparked a paradigm shift toward safer isoform-specific molecules. With pervasive roles in aggressive diseases, there remains a need for novel approaches to target these enzymes. Herein, we report the discovery of YSR734, a first-in-class covalent HDACi, with a 2-aminobenzanilide Zn2+ chelate and a pentafluorobenzenesulfonamide electrophile. This class I selective proof of concept modified HDAC2Cys274 (catalytic domain), with nM potency against HDAC1–3, sub-μM activity in MV4–11 cells, and limited cytotoxicity in MRC-9 fibroblasts. In C2C12 myoblasts, YSR734 activated muscle-specific biomarkers myogenin/Cav3, causing potent differentiation into myotubes (applications in Duchenne Muscular Dystrophy). Current efforts are focused on improving in vivo ADME toward a preclinical covalent HDACi.

Covalent targeting of non-cysteine residues in PI4KIIIβ

Brett Cosgrove, Emma K. Grant, Sophie Bertrand, Kenneth D. Down, Don O. Somers, John P. Evans d, Nicholas C. O. Tomkinson and Michael D. Barker

RSC Chem. Biol., 2023, 4, 1111-1122

DOI: 10.1039/D3CB00142C 

The synthesis and characterisation of fluorosulfate covalent inhibitors of the lipid kinase PI4KIIIβ is described. The conserved lysine residue located within the ATP binding site was targeted, and optimised compounds based upon reversible inhibitors with good activity and physicochemical profile showed strong reversible interactions and slow onset times for the covalent inhibition, resulting in an excellent selectivity profile for the lipid kinase target. X-Ray crystallography demonstrated a distal tyrosine residue could also be targeted using a fluorosulfate strategy. Combination of this knowledge showed that a dual covalent inhibitor could be developed which reveals potential in addressing the challenges associated with drug resistant mutations.

Covalent PROTAC design method based on a sulfonyl pyridone probe

Qinhong Luo, Yaqi Wang,   Zhanfeng Hou,  Huiting Liang,  Licheng Tu,  Yun Xing, Chuan Wan, Jianbo Liu,  Rui Wang, Lizhi Zhu, Wei Han,   Jianlong Wu,  Fei Lu,   Feng Yin   and  Zigang Li 

Chem. Commun., 2024

DOI: 10.1039/D3CC05127G

Covalent proteolysis-targeting chimeras (PROTACs) offer enhanced selectivity, prolonged action, and increased efficacy against challenging target proteins. The conventional approach relies on covalent ligands, but our study presents an innovative method employing an N-sulfonyl pyridone warhead to selectively target tyrosine (Tyr) residues. The von Hippel–Lindau (VHL) moiety is transferred from the warhead to the exposed Tyr, allowing us to design a STING degrader (DC50 0.53 μM, Dmax 56.65%). This approach showcases the potential of nucleophilic amino acid labeling probes, particularly for proteins lacking easily accessible cysteine residues, opening new possibilities for covalent PROTAC design and targeted protein degradation therapies.