Dichloro Butenediamides as Irreversible Site-Selective Protein Conjugation Reagent

Dr. Victor Laserna, Dr. Daniel Abegg, Cláudia F. Afonso, Dr. Esther M. Martin, Dr. Alexander Adibekian, Dr. Peter Ravn, Dr. Francisco Corzana, Dr. Gonçalo J. L. Bernardes

Angew. Chem. Int. Ed. 2021

https://doi.org/10.1002/anie.202108791

We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation.

Modeling the Binding and Conformational Energetics of a Targeted Covalent Inhibitor to Bruton’s Tyrosine Kinase [@RowleyGroup]

Ernest Awoonor-Williams and Christopher N. Rowley

Journal of Chemical Information and Modeling 2021

DOI: 10.1021/acs.jcim.1c00897

Targeted covalent inhibitors (TCIs) bind to their targets in both covalent and noncovalent modes, providing exceptionally high affinity and selectivity. These inhibitors have been effectively employed as inhibitors of protein kinases, with Taunton and coworkers (Nat. Chem. Biol.2015,11, 525–531) reporting a notable example of a TCI with a cyanoacrylamide warhead that forms a covalent thioether linkage to an active-site cysteine (Cys481) of Bruton’s tyrosine kinase (BTK). The specific mechanism of the binding and the relative importance of the covalent and noncovalent interactions is difficult to determine experimentally, and established simulation methods for calculating the absolute binding affinity of an inhibitor cannot describe the covalent bond-forming steps. Here, an integrated approach using alchemical free-energy perturbation and QM/MM molecular dynamics methods was employed to model the complete Gibbs energy profile for the covalent inhibition of BTK by a cyanoacrylamide TCI. These calculations provide a rigorous and complete absolute Gibbs energy profile of the covalent modification binding process. Following a classic thiol-Michael addition mechanism, the target cysteine is deprotonated to form a nucleophilic thiolate, which then undergoes a facile conjugate addition to the electrophilic functional group to form a bond with the noncovalently bound ligand. This model predicts that the formation of the covalent linkage is highly exergonic relative to the noncovalent binding alone. Nevertheless, noncovalent interactions between the ligand and individual amino acid residues in the binding pocket of the enzyme are also essential for ligand binding, particularly van der Waals dispersion forces, which have a larger contribution to the binding energy than the covalent component in absolute terms. This model also shows that the mechanism of covalent modification of a protein occurs through a complex series of steps and that entropy, conformational flexibility, noncovalent interactions, and the formation of covalent linkage are all significant factors in the ultimate binding affinity of a covalent drug to its target.

Methylglyoxal Forms Diverse Mercaptomethylimidazole Crosslinks with Thiol and Guanidine Pairs in Endogenous Metabolites and Proteins

 John S. Coukos and  Raymond E. Moellering*

ACS Chem. Biol. 2021

https://doi.org/10.1021/acschembio.1c00553

Methylglyoxal (MGO) is a reactive byproduct formed by several metabolic precursors, the most notable being triosephosphates in glycolysis. While many MGO-mediated adducts have been described, the reactivity and specific biomolecular targets of MGO remain incompletely mapped. Based on our recent discovery that MGO can form stable mercaptomethylimidazole crosslinks between cysteine and arginine (MICA) in proteins, we hypothesized that MGO may participate in myriad reactions with biologically relevant guanidines and thiols in proteins, metabolites, and perhaps other biomolecules. Herein, we performed steady-state and kinetic analyses of MGO reactivity with several model thiols, guanidines, and biguanide drugs to establish the plausible and prevalent adducts formed by MGO in proteins, peptides, and abundant cellular metabolites. We identified several novel, stable MICA metabolites that form in vitro and in cells, as well as a novel intermolecular post-translational MICA modification of surface cysteines in proteins. These data confirm that kinetic trapping of free MGO by thiols occurs rapidly and can decrease formation of more stable imidazolone (MG-H1) arginine adducts. However, reversible hemithioacetal adducts can go on to form stable MICA modifications in an inter- and intramolecular fashion with abundant or proximal guanidines, respectively. Finally, we discovered that intracellular MICA-glutathione metabolites are recognized and exported by the efflux pump MRP1, providing a parallel and perhaps complementary pathway for MGO detoxification working alongside the glyoxalase pathway. These data provide new insights into the plausible reactions involving MGO in cells and tissues, as well as several new molecular species in proteins and metabolites for further study.

Structure-based classification predicts drug response in EGFR-mutant NSCLC

Jacqulyne P. Robichaux, Xiuning Le, R. S. K. Vijayan, J. Kevin Hicks, Simon Heeke, Yasir Y. Elamin, Heather Y. Lin, Hibiki Udagawa, Ferdinandos Skoulidis, Hai Tran, Susan Varghese, Junqin He, Fahao Zhang, Monique B. Nilsson, Lemei Hu, Alissa Poteete, Waree Rinsurongkawong, Xiaoshan Zhang, Chenghui Ren, Xiaoke Liu, Lingzhi Hong, Jianjun Zhang, Lixia Diao, Russell Madison, Alexa B. Schrock, Jennifer Saam, Victoria Raymond, Bingliang Fang, Jing Wang, Min Jin Ha, Jason B. Cross, Jhanelle E. Gray & John V. Heymach 

Nature (2021). 

https://doi.org/10.1038/s41586-021-03898-1

Epidermal growth factor receptor (EGFR) mutations typically occur in exons 18–21 and are established driver mutations in non-small cell lung cancer (NSCLC)1,2,3. Targeted therapies are approved for patients with ‘classical’ mutations and a small number of other mutations4,5,6. However, effective therapies have not been identified for additional EGFR mutations. Furthermore, the frequency and effects of atypical EGFR mutations on drug sensitivity are unknown1,3,7,8,9,10. Here we characterize the mutational landscape in 16,715 patients with EGFR-mutant NSCLC, and establish the structure–function relationship of EGFR mutations on drug sensitivity. We found that EGFR mutations can be separated into four distinct subgroups on the basis of sensitivity and structural changes that retrospectively predict patient outcomes following treatment with EGFR inhibitors better than traditional exon-based groups. Together, these data delineate a structure-based approach for defining functional groups of EGFR mutations that can effectively guide treatment and clinical trial choices for patients with EGFR-mutant NSCLC and suggest that a structure–function-based approach may improve the prediction of drug sensitivity to targeted therapies in oncogenes with diverse mutations.

Chemoselective cysteine or disulfide modification via single atom substitution in chloromethyl acryl reagents [@WeilTanja]

Lujuan Xu,  Maria J. S. A. Silva,  Pedro M. P. Gois, Seah Ling Kuan, and  Tanja Weil 

Chemical Science, 2021

https://doi.org/10.1039/D1SC03250J

The development of bioconjugation chemistry has enabled the combination of various synthetic functionalities to proteins, giving rise to new classes of protein conjugates with functions well beyond what Nature can provide. Despite the progress in bioconjugation chemistry, there are no reagents developed to date where the reactivity can be tuned in a user-defined fashion to address different amino acid residues in proteins. Here, we report that 2-chloromethyl acryl reagents can serve as a simple yet versatile platform for selective protein modification at cysteine or disulfide sites by tuning their inherent electronic properties through the amide or ester linkage. Specifically, the 2-chloromethyl derivatives (acrylamide or acrylate) can be obtained via a simple and easily implemented one-pot reaction based on the coupling reaction between commercially available starting materials with different end-group functionalities (amino group or hydroxyl group). 2-Chloromethyl acrylamide reagents with an amide linkage favor selective modification at the cysteine site with fast reaction kinetics and near quantitative conversations. In contrast, 2-chloromethyl acrylate reagents bearing an ester linkage can undergo two successive Michael reactions, allowing the selective modification of disulfides bonds with high labeling efficiency and good conjugate stability.

Acetylation of the Catalytic Lysine Inhibits Kinase Activity in PI3Kδ

• Julie C. L. Fournier
, 
• John P. Evans
, 
• Francesca Zappacosta
, 
• Daniel A. Thomas
, 
• Vipulkumar K. Patel*
, 
• Gemma V. White
, 
• Sebastien Campos
, and 
• Nicholas C. O. Tomkinson
• 
  
  ACS Chem. Biol. 2021
  https://doi.org/10.1021/acschembio.1c00225
  
  
  Covalent inhibition is a powerful strategy to develop potent and selective small molecule kinase inhibitors. Targeting the conserved catalytic lysine is an attractive method for selective kinase inactivation. We have developed novel, selective inhibitors of phosphoinositide 3-kinase δ (PI3Kδ) which acylate the catalytic lysine, Lys779, using activated esters as the reactive electrophiles. The acylating agents were prepared by adding the activated ester motif to a known selective dihydroisobenzofuran PI3Kδ inhibitor. Three esters were designed, including an acetate ester which was the smallest lysine modification evaluated in this work. Covalent binding to the enzyme was characterized by intact protein mass spectrometry of the PI3Kδ-ester adducts. An enzymatic digest coupled with tandem mass spectrometry identified Lys779 as the covalent binding site, and a biochemical activity assay confirmed that PI3Kδ inhibition was a direct result of covalent lysine acylation. These results indicate that a simple chemical modification such as lysine acetylation is sufficient to inhibit kinase activity. The selectivity of the compounds was evaluated against lipid kinases in cell lysates using a chemoproteomic binding assay. Due to the conserved nature of the catalytic lysine across the kinome, we believe the covalent inhibition strategy presented here could be applicable to a broad range of clinically relevant targets.

A proteome-wide atlas of lysine-reactive chemistry [@Abbasov_Cornell]

Mikail E. Abbasov, Madeline E. Kavanagh, Taka-Aki Ichu, Michael R. Lazear, Yongfeng Tao, Vincent M. Crowley, Christopher W. am Ende, Stephan M. Hacker, Jordan Ho, Melissa M. Dix, Radu Suciu, Matthew M. Hayward, Laura L. Kiessling & Benjamin F. Cravatt 

Nature Chemistry, 2021

https://doi.org/10.1038/s41557-021-00765-4

Recent advances in chemical proteomics have begun to characterize the reactivity and ligandability of lysines on a global scale. Yet, only a limited diversity of aminophilic electrophiles have been evaluated for interactions with the lysine proteome. Here, we report an in-depth profiling of >30 uncharted aminophilic chemotypes that greatly expands the content of ligandable lysines in human proteins. Aminophilic electrophiles showed disparate proteomic reactivities that range from selective interactions with a handful of lysines to, for a set of dicarboxaldehyde fragments, remarkably broad engagement of the covalent small-molecule–lysine interactions captured by the entire library. We used these latter ‘scout’ electrophiles to efficiently map ligandable lysines in primary human immune cells under stimulatory conditions. Finally, we show that aminophilic compounds perturb diverse biochemical functions through site-selective modification of lysines in proteins, including protein–RNA interactions implicated in innate immune responses. These findings support the broad potential of covalent chemistry for targeting functional lysines in the human proteome.

Dichloro Butendiamides as Irreversible Site-Selective Protein Conjugation Reagent [@gbernardes_chem]

Victor Laserna, Daniel Abegg, Cláudia Afonso,Esther Martin,Alexander Adibekian, Peter Ravn, Francisco Corzana, Gonçalo J. L. Bernardes

Angewandte Chemie International Edition 2021

https://doi.org/10.1002/anie.202108791

We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to predominantly used maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization reaction occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates makes this strategy an important alternative to maleimides in the bioconjugation toolbox.