Activity-Based Acylome Profiling with N-(Cyanomethyl)-N-(phenylsulfonyl)amides for Targeted Lysine Acylation and Post-Translational Control of Protein Function in Cells

Elizabeth M. Ryan, Michael A. Norinskiy, Amy K. Bracken, Emma E. Lueders, Xueer Chen, Qin Fu, Elizabeth T. Anderson, Sheng Zhang, and Mikail E. Abbasov

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c09073

Lysine acylations are ubiquitous and structurally diverse post-translational modifications that vastly expand the functional heterogeneity of the human proteome. Hence, the targeted acylation of lysine residues has emerged as a strategic approach to exert biomimetic control over the protein function. However, existing strategies for targeted lysine acylation in cells often rely on genetic intervention, recruitment of endogenous acylation machinery, or nonspecific acylating agents and lack methods to quantify the magnitude of specific acylations on a global level. In this study, we develop activity-based acylome profiling (ABAP), a chemoproteomic strategy that exploits elaborate N-(cyanomethyl)-N-(phenylsulfonyl)amides and lysine-centric probes for site-specific introduction and proteome-wide mapping of posttranslational lysine acylations in human cells. Harnessing this framework, we quantify various artificial acylations and rediscover numerous endogenous lysine acylations. We validate site-specific acetylation of target lysines and establish a structure–activity relationship for N-(cyanomethyl)-N-(phenylsulfonyl)amides in proteins from diverse structural and functional classes. We identify paralog-selective chemical probes that acetylate conserved lysines within interferon-stimulated antiviral RNA-binding proteins, generating de novo proteoforms with obstructed RNA interactions. We further demonstrate that targeted acetylation of a key enzyme in retinoid metabolism engenders a proteoform with a conformational change in the protein structure, leading to a gain-of-function phenotype and reduced drug potency. These findings underscore the versatility of our strategy in biomimetic control over protein function through targeted delivery and global profiling of endogenous and artificial lysine acylations, potentially advancing therapeutic modalities and our understanding of biological processes orchestrated by these post-translational modifications.

Isocyanides inhibit bacterial pathogens by covalent targeting of essential metabolic enzymes

Alexandra Geißler, Howard Junca , Andreas M. Kany , Lena J. Daumann, Anna K. H. Hirsch  Dietmar H. Pieper b and Stephan A. Sieber 

Chem. Sci., 2024, 15, 11946-11955

 https://doi.org/10.1039/D4SC01940G

Isonitrile natural products, also known as isocyanides, demonstrate potent antimicrobial activities, yet our understanding of their molecular targets remains limited. Here, we focus on the so far neglected group of monoisonitriles to gain further insights into their antimicrobial mode of action (MoA). Screening a focused monoisonitrile library revealed a potent S. aureus growth inhibitor with a different MoA compared to previously described isonitrile antibiotics. Chemical proteomics via competitive cysteine reactivity profiling, uncovered covalent modifications of two essential metabolic enzymes involved in the fatty acid biosynthetic process (FabF) and the hexosamine pathway (GlmS) at their active site cysteines. In-depth studies with the recombinant enzymes demonstrated concentration-dependent labeling, covalent binding to the catalytic site and corresponding functional inhibition by the isocyanide. Thermal proteome profiling and full proteome studies of compound-treated S. aureus further highlighted the destabilization and dysregulation of proteins related to the targeted pathways. Cytotoxicity and the inhibition of cytochrome P450 enzymes require optimization of the hit molecule prior to therapeutic application. The here described novel, covalent isocyanide MoA highlights the versatility of the functional group, making it a useful tool and out-of-the-box starting point for the development of innovative antibiotics.

Peptide and Protein Cysteine Modification Enabled by Hydrosulfuration of Ynamide

Changliu Wang, Zhenguang Zhao, Reem Ghadir, Dechun Yang, Zhenjia Zhang, Zhe Ding, Yuan Cao, Yuqing Li, Rosi Fassler, Dana Reichmann, Yujie Zhang, Yongli Zhao, Can Liu, Xiaobao Bi, Norman Metanis, and Junfeng Zhao

ACS Central Science 2024 10 (9), 1742-1754

DOI: 10.1021/acscentsci.4c01148

Efficient functionalization of peptides and proteins has widespread applications in chemical biology and drug discovery. However, the chemoselective and site-selective modification of proteins remains a daunting task. Herein, a highly efficient chemo-, regio-, and stereoselective hydrosulfuration of ynamide was identified as an efficient method for the precise modification of peptides and proteins by uniquely targeting the thiol group of cysteine (Cys) residues. This novel method could be facilely operated in aqueous buffer and was fully compatible with a wide range of proteins, including small model proteins and large full-length antibodies, without compromising their integrity and functions. Importantly, this reaction provides the Z-isomer of the corresponding conjugates exclusively with superior stability, offering a precise approach to peptide and protein therapeutics. The potential application of this method in peptide and protein chemical biology was further exemplified by Cys-bioconjugation with a variety of ynamide-bearing functional molecules such as small molecule drugs, fluorescent/affinity tags, and PEG polymers. It also proved efficient in redox proteomic analysis through Cys-alkenylation. Overall, this study provides a novel bioorthogonal tool for Cys-specific functionalization, which will find broad applications in the synthesis of peptide/protein conjugates.

An allosteric cyclin E-CDK2 site mapped by paralog hopping with covalent probes

Yuanjin Zhang, Zhonglin Liu, Marscha Hirschi, Oleg Brodsky, Eric Johnson, Sang Joon Won, Asako Nagata, Divya Bezwada, Matthew D. Petroski, Jaimeen D. Majmudar, Sherry Niessen, Todd VanArsdale, Adam M. Gilbert, Matthew M. Hayward, Al E. Stewart, Andrew R. Nager, Bruno Melillo & Benjamin F. Cravatt 

Nat Chem Biol, 2024 

https://doi.org/10.1038/s41589-024-01738-7

More than half of the ~20,000 protein-encoding human genes have paralogs. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to subsets of paralogous proteins. Here we explore whether such covalent compound–cysteine interactions can be used to discover ligandable pockets in paralogs lacking the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we substituted the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide–CCNE1-N112C interaction into in vitro NanoBRET (bioluminescence resonance energy transfer) and in cellulo activity-based protein profiling assays capable of identifying compounds that reversibly inhibit both the N112C mutant and wild-type CCNE1:CDK2 (cyclin-dependent kinase 2) complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings, thus, show how electrophile–cysteine interactions mapped by chemical proteomics can extend the understanding of protein ligandability beyond covalent chemistry.

Nuclear Receptors: A new mode of inhibition

1. Andrew D Huber, 

1. Taosheng Chen
 

eLife 2024, 13:e101446.

https://doi.org/10.7554/eLife.101446

The article presents the discussion on interaction of covalent inhibitors GW9662 and T0070907 with the peroxisome proliferator-activated receptor gamma (PPARγ), revealing new insights into how these compounds influence receptor activity. Topics include the role of PPARγ in regulating fatty tissue and glucose levels; and the effects of covalent inhibitors on PPARγ's ligand-binding site and its interaction with co-activators and co-repressors.

Selective Covalent Inhibiting JNK3 by Small Molecules for Parkinson's Diseases

Liang Ouyang, Wen Shuai, Panpan Yang, Huan Xiao, Yumeng Zhu, Faqian Bu, Aoxue Wang, Qiu Sun, Guan Wang

Angewandte Chemie 2024 e202411037

https://doi.org/10.1002/ange.202411037

c-Jun N-terminal kinases (JNKs) including JNK1/2/3 are key members of mitogen-activated protein kinase family. Wherein JNK3 is specifically expressed in brain and emerges as therapeutic target, especially for neurodegenerative diseases. However, developing JNK3 selective inhibitors as chemical probes to investigate its therapeutic potential in diseases remains challenging. Here, we adopted the covalent strategy for identifying JNK3-selective covalent inhibitorJC16I, with high inhibitory activity against JNK3. Despite targeting a conserved cysteine the vicinity of ATP pocket in JNK family, JC16I exerted a greater than 160-fold selectivity for JNK3 over JNK1/2. Importantly, even at low concentration, JC16I showed enhanced and long-lasting inhibition against cellular JNK3. In addition, its alkyne-containing probe JC-P1 could label JNK3 in SH-SY5Y cell lysate and living cells, with goodproteome-wide selectivity. Furthermore, JC16I selectively suppressed the abnormal activation of JNK3 signaling and sufficiently exhibited neuroprotective effect in Parkinson's diseases (PD) models. Overall, our findings highlight the potential of developing isoform-selective and cell-active JNK3 inhibitors by covalent drug design strategy targeting a conserved cysteine. This work not only provides a valuable chemical probe for JNK3-targeted investigations in vitro and in vivo but also opens new avenues for the treatment of PD.

Development of ketalized unsaturated saccharides as multifunctional cysteine-targeting covalent warheads

Dong, S., Huang, H., Li, J. et al.

Commun Chem 7, 201 (2024). 

https://doi.org/10.1038/s42004-024-01279-z

Multi-functional cysteine-targeting covalent warheads possess significant therapeutic potential in medicinal chemistry and chemical biology. Herein, we present novel unsaturated and asymmetric ketone (oxazolinosene) scaffolds that selectively conjugate cysteine residues of peptides and bovine serum albumin under normal physiological conditions. This unsaturated saccharide depletes GSH in NCI-H1299 cells, leading to anti-tumor effects in vitro. The acetyl group of the ketal moiety on the saccharide ring can be converted to other carboxylic acids in a one-pot synthesis. In this way, the loaded acid can be click-released during cysteine conjugation, making the oxazolinosene a potential multifunctional therapeutic agent. The reaction kinetic model for oxazolinosene conjugation to GSH is well established and was used to evaluate oxazolinosene reactivity. The aforementioned oxazolinosenes were stereoselectively synthesized via a one-step reaction of nitriles with saccharides and conveniently converted into a series of α, β-unsaturated ketone N-glycosides as prevalent synthetic building blocks. The reaction mechanisms of oxazolinosene synthesis were investigated through calculations and validated with control experiments. Overall, these oxazolinosenes can be easily synthesized and developed as cysteine-targeted covalent warheads carrying useful click-releasing groups.

From Mechanism-Based Retaining Glycosidase Inhibitors to Activity-Based Glycosidase Profiling

 Marta Artola, Johannes M. F. G. Aerts, Gijsbert A. van der Marel, Carme Rovira, Jeroen D. C. Codée, Gideon J. Davies, and Herman S. Overkleeft

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c08840

Activity-based protein profiling (ABPP) is an effective technology for the identification and functional annotation of enzymes in complex biological samples. ABP designs are normally directed to an enzyme active site nucleophile, and within the field of Carbohydrate-Active Enzymes (CAZymes), ABPP has been most successful for those enzymes that feature such a residue: retaining glycosidases (GHs). Several mechanism-based covalent and irreversible retaining GH inhibitors have emerged over the past sixty years. ABP designs based on these inhibitor chemistries appeared since the turn of the millennium, and we contributed to the field by designing a suite of retaining GH ABPs modeled on the structure and mode of action of the natural product, cyclophellitol. These ABPs enable the study of both exo- and endo-acting retaining GHs in human health and disease, for instance in genetic metabolic disorders in which retaining GHs are deficient. They are also finding increasing use in the study of GHs in gut microbiota and environmental microorganisms, both in the context of drug (de)toxification in the gut and that of biomass polysaccharide processing for future sustainable energy and chemistries. This account comprises the authors’ view on the history of mechanism-based retaining GH inhibitor design and discovery, on how these inhibitors served as blueprints for retaining GH ABP design, and on some current and future developments on how cyclophellitol-based ABPs may drive the discovery of retaining GHs and their inhibitors.

SuFEx Chemistry Enables Covalent Assembly of a 280-kDa 18-Subunit Pore-Forming Complex

Lee Schnaider, Sophia Tan, Pratik R. Singh, Floriana Capuano, Alistair J. Scott, Richard Hambley, Lei Lu, Hyunjun Yang, E. Jayne Wallace, Hyunil Jo, and William F. DeGrado

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c07920

Proximity-enhanced chemical cross-linking is an invaluable tool for probing protein–protein interactions and enhancing the potency of potential peptide and protein drugs. Here, we extend this approach to covalently stabilize large macromolecular assemblies. We used SuFEx chemistry to covalently stabilize an 18-subunit pore-forming complex, CsgG:CsgF, consisting of nine CsgG membrane protein subunits that noncovalently associate with nine CsgF peptides. Derivatives of the CsgG:CsgF pore have been used for DNA sequencing, which places high demands on the structural stability and homogeneity of the complex. To increase the robustness of the pore, we designed and synthesized derivatives of CsgF-bearing sulfonyl fluorides, which react with CsgG in very high yield to form a covalently stabilized CsgG:CsgF complex. The resulting pores formed highly homogeneous channels when added to artificial membranes. The high yield and rapid reaction rate of the SuFEx reaction prompted molecular dynamics simulations, which revealed that the SO2F groups in the initially formed complex are poised for nucleophilic reaction with a targeted Tyr. These results demonstrate the utility of SuFEx chemistry to structurally stabilize very large (here, 280 kDa) assemblies.