Mutate and Conjugate: A Method to Enable Rapid In-Cell Target Validation

Thomas, A.; Serafini, M.; Grant, E.; Coombs, E.; Bluck, J.; Schiedel, M.; McDonough, M.; Reynolds, J.; Lee, B.; Platt, M.; Sharlandjieva, V.; Biggin, P.; Duarte, F.; Milne, T.; Bush, J.; Conway, S. 

ChemRxiv 2023. 

https://doi.org/10.26434/chemrxiv-2023-8qnvr

Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain- containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having minimal effect on BRD4(1) function. We then screen a focused, KAc mimic-containing fragment set, and a diverse fragment library against the mutant and wild-type proteins, and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutant and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for non-covalent ligand development.

 

Cell Surface Labeling and Detection of Protein Tyrosine Kinase 7 via Covalent Aptamers

Savannah Albright, Mary Cacace, Yaniv Tivon, and Alexander Deiters Journal of the American Chemical Society 2023 DOI: 10.1021/jacs.3c02752

Covalent aptamers are novel biochemical tools for fast and selective transfer of labels to target proteins. Equipped with cleavable electrophiles, these nucleic acid probes enable the installation of functional handles onto native proteins. The high affinity and specificity with which aptamers bind their selected targets allows for quick, covalent labeling that can compete with nuclease-mediated degradation. Here, we introduce the first application of covalent aptamers to modify a specific cell surface protein through proximity-driven label transfer. We targeted protein tyrosine kinase 7 (PTK7), a prominent cancer marker, and demonstrated aptamer-mediated biotin transfer to specific lysine residues on the extracellular domain of the protein. This allowed for tracking of PTK7 expression, localization, and cellular internalization. These studies validate the programmability of covalent aptamers and highlight their applicability in a cellular context, including protein and small molecule delivery

Proteome-wide structural analysis identifies warhead-and coverage-specific biases in cysteine-focused chemoproteomics

Matthew E H White, Jesús Gil, Edward W Tate

https://doi.org/10.1016/j.chembiol.2023.06.021

Covalent drug discovery has undergone a resurgence over the past two decades and reactive cysteine profiling has emerged in parallel as a platform for ligand discovery through on- and off-target profiling; however, the scope of this approach has not been fully explored at the whole-proteome level. We combined AlphaFold2-predicted side-chain accessibilities for >95% of the human proteome with a meta-analysis of eighteen public cysteine profiling datasets, totaling 44,187 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further showed that hit elaboration and optimization drives increased bias against buried cysteine residues. Based on these data, we suggest that current profiling approaches cover a small proportion of potential ligandable cysteine residues and propose future directions for increasing coverage, focusing on high-priority residues and depth. All analysis and produced resources are freely available and extendable to other reactive amino acids.

Direct mapping of ligandable tyrosines and lysines in cells with chiral sulfonyl fluoride probes

Ying Chen, Gregory B. Craven, Roarke A. Kamber, Adolfo Cuesta, Serhii Zhersh, Yurii S. Moroz, Michael C. Bassik & Jack Taunton 

Nature Chemistry 2023

https://doi.org/10.1038/s41557-023-01281-3

Advances in chemoproteomic technology have revealed covalent interactions between small molecules and protein nucleophiles, primarily cysteine, on a proteome-wide scale. Most chemoproteomic screening approaches are indirect, relying on competition between electrophilic fragments and a minimalist electrophilic probe with inherently limited proteome coverage. Here we develop a chemoproteomic platform for direct electrophile-site identification based on enantiomeric pairs of clickable arylsulfonyl fluoride probes. Using stereoselective site modification as a proxy for ligandability in intact cells, we identify 634 tyrosines and lysines within functionally diverse protein sites, liganded by structurally diverse probes. Among multiple validated sites, we discover a chiral probe that modifies Y228 in the MYC binding site of the epigenetic regulator WDR5, as revealed by a high-resolution crystal structure. A distinct chiral probe stimulates tumour cell phagocytosis by covalently modifying Y387 in the recently discovered immuno-oncology target APMAP. Our work provides a deep resource of ligandable tyrosines and lysines for the development of covalent chemical probes.

N-Acryloylindole-alkyne (NAIA) enables imaging and profiling new ligandable cysteines and oxidized thiols by chemoproteomics

Nat Commun 14, 3564 (2023). 

https://doi.org/10.1038/s41467-023-39268-w

Cysteine has been exploited as the binding site of covalent drugs. Its high sensitivity to oxidation is also important for regulating cellular processes. To identify new ligandable cysteines which can be hotspots for therapy and to better study cysteine oxidations, we develop cysteine-reactive probes, N-acryloylindole-alkynes (NAIAs), which have superior cysteine reactivity owing to delocalization of π electrons of the acrylamide warhead over the whole indole scaffold. This allows NAIAs to probe functional cysteines more effectively than conventional iodoacetamide-alkyne, and to image oxidized thiols by confocal fluorescence microscopy. In mass spectrometry experiments, NAIAs successfully capture new oxidized cysteines, as well as a new pool of ligandable cysteines and proteins. Competitive activity-based protein profiling experiments further demonstrate the ability of NAIA to discover lead compounds targeting these cysteines and proteins. We show the development of NAIAs with activated acrylamide for advancing proteome-wide profiling and imaging ligandable cysteines and oxidized thiols.

Expanding the Chemistry of Dihaloacetamides as Tunable Electrophiles for Reversible Covalent Targeting of Cysteines

Daiki Yamane, Ryo Tetsukawa, Naoki Zenmyo, Kaori Tabata, Yuya Yoshida, Naoya Matsunaga, Naoya Shindo, and Akio Ojida

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.3c00737

The choice of an appropriate electrophile is crucial in the design of targeted covalent inhibitors (TCIs). In this report, we systematically investigated the glutathione (GSH) reactivity of various haloacetamides and the aqueous stability of their thiol adducts. Our findings revealed that dihaloacetamides cover a broad range of GSH reactivity depending on the combination of the halogen atoms and the structure of the amine scaffold. Among the dihaloacetamides, dichloroacetamide (DCA) exhibited slightly lower GSH reactivity than chlorofluoroacetamide (CFA). The DCA–thiol adduct is readily hydrolyzed under aqueous conditions, but it can stably exist in the solvent-sequestered binding pocket of the protein. These reactivity profiles of DCA were successfully exploited in the design of TCIs targeting noncatalytic cysteines of KRASG12C and EGFRL858R/T790M. These inhibitors exhibited strong antiproliferative activities against cancer cells. Our findings provide valuable insights for designing dihaloacetamide-based reversible covalent inhibitors.