Thursday, November 25, 2021

Site-Specific Labeling of Endogenous Proteins Using CoLDR Chemistry

Rambabu N. Reddi, Adi Rogel, Efrat Resnick, Ronen Gabizon, Pragati Kishore Prasad, Neta Gurwicz, Haim Barr, Ziv Shulman, and Nir London

Journal of the American Chemical Society 2021

DOI: 10.1021/jacs.1c06167

Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive “turn-on” fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.

Wednesday, November 24, 2021

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway [@AdibekianG]

Daniel Abegg, Martin Tomanik, Nan Qiu, Dany Pechalrieu, Anton Shuster, Bruno Commare, Antonio Togni, Seth B. Herzon, and Alexander Adibekian

Journal of the American Chemical Society 2021

DOI: 10.1021/jacs.1c09724

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine “signature” of probe 7 constitutes an additional advantage resulting in a more confident adduct–amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (−)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

Improved Electrophile Design for Exquisite Covalent Molecule Selectivity [@BalynZaro]

José L. Montaño, Brian J. Wang, Regan F. Volk, Virginia G. Garda, Balyn W. Zaro*

ChemRxiv, 2021

D O I: 10.26434/chemrxiv-2021-67z8j-v2 [opens in a new tab]

Covalent inhibitors continue to show therapeutic promise. However, off-target reactivity challenges the field. Extensive efforts have been exerted to solve this issue by varying the reactivity attributes of electrophilic warheads, with features such as reversibility or metabolic vulnerability. Here we report the development of a new approach to increase the selectivity of covalent probes and small molecule inhibitors that is independent of warhead reactivity features and can be used in concert with already-existing methods. Using the Bruton’s Tyrosine Kinase (BTK) inhibitor Ibrutinib scaffold for our proof-of-concept, we reasoned that increasing the steric bulk of fumarate-based electrophiles on Ibrutinib should improve selectivity via the steric exclusion of off-targets but ideally retain rates of cysteine reactivity comparable to that of an acrylamide. Using chemical proteomic techniques, we demonstrate that elaboration of the electrophile to a tert-Butyl (t-Bu) fumarate ester significantly decreases time-dependent off-target reactivity and abolishes time-independent off-target reactivity but retains BTK target engagement. While an alkyne-bearing probe analog of Ibrutinib has 247 protein targets, our t-Bu fumarate Ibrutinib probe analog has only 7 protein targets. Of these 7 targets, BTK is the only time-independent target. This 2-order-of-magnitude increase in selectivity is also conferred to the t-Bu inhibitor itself. By shotgun proteomics, we investigated the consequences of treatment with Ibrutinib and our t-Bu analog and discovered that only 8 proteins are downregulated in response to treatment with the t-Bu analog compared to 107 with Ibrutinib. Of these 8 proteins, 7 are also downregulated by Ibrutinib and a majority of these targets are associated with BTK biology. Taken together, these findings reveal a previously-unappreciated opportunity to increase cysteine-reactive covalent inhibitor selectivity through electrophilic structure optimization.

Friday, November 19, 2021

Metabolically Activated Proteostasis Regulators Protect against Glutamate Toxicity by Activating NRF2

Jessica D. Rosarda, Kelsey R. Baron, Kayla Nutsch, Gabriel M. Kline, Caroline Stanton, Jeffery W. Kelly, Michael J. Bollong, and R. Luke Wiseman

ACS Chemical Biology 2021

DOI: 10.1021/acschembio.1c00810

The extracellular accumulation of glutamate is a pathologic hallmark of numerous neurodegenerative diseases including ischemic stroke and Alzheimer’s disease. At high extracellular concentrations, glutamate causes neuronal damage by promoting oxidative stress, which can lead to cellular death. This has led to significant interest in developing pharmacologic approaches to mitigate the oxidative toxicity caused by high levels of glutamate. Here, we show that the small molecule proteostasis regulator AA147 protects against glutamate-induced cell death in a neuronal-derived cell culture model. While originally developed as an activator of the activating transcription factor 6 (ATF6) arm of the unfolded protein response, this AA147-dependent protection against glutamate toxicity is primarily mediated through activation of the NRF2-regulated oxidative stress response. We demonstrate that AA147 activates NRF2 selectively in neuronal-derived cells through a mechanism involving metabolic activation to a reactive electrophile and covalent modification of KEAP1─a mechanism analogous to that involved in the AA147-dependent activation of ATF6. These results define the potential for AA147 to protect against glutamate-induced oxidative toxicity and highlight the potential for metabolically activated proteostasis regulators like AA147 to activate both protective ATF6 and NRF2 stress-responsive signaling pathways to mitigate oxidative damage associated with diverse neurologic diseases.

Monday, November 15, 2021

Tunable Amine-Reactive Electrophiles for Selective Profiling of Lysine [@Keribackus, @MonikaRaj_lab ]

Monika Raj, Kuei-Chien Tang,Jian Cao, Lisa M. Boatner, Linwei Li,Jonathan Farhi, Kendall N. Houk, Jennifer Spangle, Keriann M. Backus

Angewandte Chemie, 2021

Proteome profiling by activated esters identified >9000 ligandable lysines but they are limited as covalent inhibitors due to poor hydrolytic stability. Here we report our efforts to design and discover a new series of Tunable Amine- Reactive  lEectrophiles (TAREs) for selective and robust labeling of lysine. The major challenges in developing selective covalent ligands for lysine are the high nucleophilicity of cysteines and poor hydrolytic stability. Our work circumvents these challenges by a unique design of the TAREs that form stable adducts with lysine and on reaction with cysteine generate another reactive electrophiles for lysine. We highlight that TAREs exhibit substantially high hydrolytic stability as compared to the activated esters and are non-cytotoxic thus have the potential to act as covalent ligands. We applied these alternative TAREs for the intracellular labeling of proteins, and for the selective identification of lysines in the human proteome on a global scale.

Sunday, November 14, 2021

Proximity-Dependent Labeling of Cysteines [@Pthompsn1018, @jusudeshna]

Sudeshna Sen, Nadia Sultana, Scott A. Shaffer, and Paul R. Thompson
Journal of the American Chemical Society 2021

DOI: 10.1021/jacs.1c07069

Mapping protein–protein interactions is crucial for understanding various signaling pathways in living cells, and developing new techniques for this purpose has attracted significant interest. Classic methods (e.g., the yeast two-hybrid) have been supplanted by more sophisticated chemical approaches that label proximal proteins (e.g., BioID, APEX). Herein we describe a proximity-based approach that uniquely labels cysteines. Our approach exploits the nicotinamide N-methyltransferase (NNMT)-catalyzed methylation of an alkyne-substituted 4-chloropyridine (SS6). Upon methylation of the pyridinium nitrogen, this latent electrophile diffuses out of the active site and labels proximal proteins on short time scales (≤5 min). We validated this approach by identifying known (and novel) interacting partners of protein arginine deiminase 2 (PAD2) and pyruvate dehydrogenase kinase 1 (PDK1). To our knowledge, this technology uniquely exploits a suicide substrate to label proximal cysteines in live cells.

Thursday, November 11, 2021

Mechanism-Based and Computational-Driven Covalent Drug Design [@lyna_yunluo]

Yun Lyna Luo

Journal of Chemical Information and Modeling 2021
DOI: 10.1021/acs.jcim.1c01278

Covalent drugs offer higher efficacy and longer duration of action than their noncovalent counterparts. Significant advances in computational methods for modeling covalent drugs are poised to shift the paradigm of small molecule therapeutics within the next decade. This viewpoint discusses the advantages of a two-state model for ranking reversible and irreversible covalent ligands and of more complex models for dissecting reaction mechanisms. The relation between these models highlights the complexity and diversity of covalent drug binding and provides opportunities for mechanism-based rational design.

Friday, November 5, 2021

Crystal structure of SARS-CoV-2 main protease in complex with protease inhibitor PF-07321332

Yao Zhao, Chao Fang, Qi Zhang, Ruxue Zhang, Xiangbo Zhao, Yinkai Duan, Haofeng Wang, Yan Zhu, Lu Feng, Jinyi Zhao, Maolin Shao, Xiuna Yang, Leike Zhang, Chao Peng, Kailin Yang, Dawei Ma, Zihe Rao & Haitao Yang 

Protein Cell, 2021

Thursday, November 4, 2021

Covalent Sortase A Inhibitor ML346 Prevents Staphylococcus aureus Infection of Galleria mellonella


Xiang-Na Guan,   Tao Zhang,   Teng Yang,   Ze Dong,   Song Yang, Jianhua Gan,   Le-Fu Lan  and  Cai-Guang Yang

The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation of bacterial virulence and pathogenicity. Therefore, SrtA is considered to be an ideal target for antivirulence therapy. In this study, we report that ML346, a compound with a barbituric acid and cinnamaldehyde scaffold, functions as an irreversible inhibitor of Staphylococcus aureus SrtA (SaSrtA) and Streptococcus pyogenes SrtA (SpSrtA) in vitro at low micromolar concentrations. According to our X-ray crystal structure of the SpSrtAΔN81/ML346 complex (Protein Data Bank ID: 7V6K), ML346 covalently modifies the thiol group of Cys208 in the active site of SpSrtA. Importantly, ML346 significantly attenuated the virulence phenotypes of S. aureus and exhibited inhibitory effects on Galleria mellonella larva infection caused by S. aureus. Collectively, our results indicate that ML346 has potential for development as a covalent antivirulence agent for treating S. aureus infections, including methicillin-resistant S. aureus. 

Monday, October 18, 2021

A Paal-Knorr agent for chemoproteomic profiling of targets of isoketals in cells

Min-Ran Wang, Jing-Yang He, Ji-Xiang He, Ke-Ke Liu and Jing Yang 

Chemical Science 2021 


Natural systems produce various γ-dicarbonyl-bearing compounds that can covalently modify lysine in protein targets via the classic Paal-Knorr reaction. Among them is a unique class of lipid-derived electrophiles - isoketals that exhibit high chemical reactivity and critical biological functions. However, it remains unknown about their target selectivity and profiles in complex proteomes. Here we report a Paal-Knorr agent, 4-oxonon-8-ynal (herein termed ONAyne), for surveying the reactivity and selectivity of the γ-dicarbonyl warhead in biological systems. Using an unbiased open-search strategy, we demonstrated the lysine specificity of ONAyne on a proteome-wide scale and charaterized six probe-derived modifications, including the initial pyrrole adduct and its oxidative products (i.e., lactam and hydroxylactam adducts), an enlactam adduct from dehyration of hydroxylactam, and two chemotypes formed in the presence of endogenous formaldehyde (i.e., fulvene and aldehyde adducts). Furthermore, combined with quantitative chemoproteomics in a competitive format, ONAyne permitted global, in situ, and site-specific profiling of targeted lysine residues of two specific isomers of isoketals, levuglandin (LG) D2 and E2. The functional analyses reveal that LGs-derived adduction drives inhibition of malate dehydrogenase MDH2 and exhibits a crosstalk with two epigenetic marks on histone H2B in macrophages. Our approach should be broadly useful for target profiling of bioactive γ-dicarbonyls in diverse biological contexts.

Identification of covalent inhibitors that disrupt M. tuberculosis growth by targeting multiple serine hydrolases involved in lipid metabolism

Brett M. Babin, Laura J. Keller, Yishay Pinto, Veronica L. Li, Andrew S. Eneim, Summer E. Vance, Stephanie M. Terrell, Ami S. Bhatt, Jonathan Z. Long, Matthew Bogyo

Cell Chemical Biology, 2021

The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screen a library of small molecules containing serine-reactive electrophiles and identify narrow-spectrum inhibitors of M. tuberculosis growth. Using these lead molecules, we perform competitive activity-based protein profiling and identify multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures reveal an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis reveals a path to resistance via the synthesis of mycocerates, but not through mutations to SH targets. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M. tuberculosis infections.

Sunday, October 10, 2021

Sulforaphane covalently interacts with the transglutaminase 2 cancer maintenance protein to alter its structure and suppress its activity

Rorke EA, Adhikary G, Szmacinski H, Lakowicz JR, Weber DJ, Godoy-Ruiz R, Puranik P, Keillor JW, Gates EWJ, Eckert RL. 

 Mol Carcinog. 2021

Type 2 transglutaminase (TG2) functions as an important cancer cell survival protein in a range of cancers including epidermal squamous cell carcinoma. TG2 exists in open and closed conformations each of which has a distinct and mutually exclusive activity. The closed conformation has GTP-binding/GTPase activity while the open conformation functions as a transamidase to catalyze protein-protein crosslinking. GTP-binding/GTPase activity is required for TG2 maintenance of the aggressive cancer phenotype. Thus, identifying agents that convert TG2 from the closed to the open GTP-binding/GTPase inactive conformation is an important cancer prevention/treatment strategy. Sulforaphane (SFN) is an important diet-derived cancer prevention agent that is known to possess a reactive isothiocyanate group and has potent anticancer activity. Using a biotin-tagged SFN analog (Biotin-ITC) and kinetic analysis we show that SFN covalently and irreversibly binds to recombinant TG2 to inhibit transamidase activity and shift TG2 to an open/extended conformation, leading to a partial inhibition of GTP binding. We also show that incubation of cancer cells or cancer cell extract with Biotin-ITC results in formation of a TG2/Biotin-ITC complex and that SFN treatment of cancer cells inhibits TG2 transamidase activity and shifts TG2 to an open/extended conformation. These findings identify TG2 as a direct SFN anticancer target in epidermal squamous cell carcinoma.

Friday, October 8, 2021

Inhibition of autophagy by a small molecule through covalent modification of LC3

Cheng Luo, Shijie Fan, Liyan Yue, Wei Wan, Yuanyuan Zhang, Bidong Zhang, Chinatsu Otomo, Quanfu Li, Tingting Lin, Junchi Hu, Pan Xu, Mingrui Zhu, Hongru Tao, Zhifeng Chen, Lianchun Li, Hong Ding, Zhiyi Yao, Junyan Lu, Yi Wen, Naixia Zhang, Minjia Tan, Kaixian Chen, Yuli Xie, Takanori Otomo, Bing Zhou, Hualiang Jiang, Yongjun Dang

Angew. Chem. Int. Ed. 2021

The autophagic ubiquitin-like protein LC3 functions through interactions with LC3-interaction regions (LIRs) of other autophagy proteins including autophagy receptors, which stands out as a promising protein-protein interaction (PPI) target for the intervention of autophagy. Post-translational modifications like acetylation of Lys49 on the LIR-interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering the interface. Through screening covalent compounds, we discover a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49. Activity-based protein profiling (ABPP) based evaluations reveal that a derivative molecule DC-LC3in-D5 exhibits a potent covalent reactivity and selectivity to LC3A/B in HeLa cells. DC-LC3in-D5 compromises LC3B lipidation in vitro and in HeLa cells, leading to deficiency in the formation of autophagic structures and autophagic substrate degradation. DC-LC3in-D5 could serve as a powerful tool for autophagy research as well as for therapeutic interventions.

Thursday, October 7, 2021

Characterization of an Aromatic Trifluoromethyl Ketone as a New Warhead for Covalently Reversible Kinase Inhibitor Design

Zhen Zhang, Yongjin Wang, Xiaojuan Chen, Xiaojuan Song, Zhengchao Tu, Yongheng Chen, Zhimin Zhang, Ke Ding,

Bioorganic & Medicinal Chemistry,2021, 116457,

An aromatic trifluoromethyl ketone moiety was characterized as a new warhead for covalently reversible kinase inhibitor design to target the non-catalytic cysteine residue. Potent and selective covalently reversible inhibitors of FGFR4 kinase were successfully designed and synthesized by utilizing this new warhead. The binding mode of a representative inhibitor was fully characterized by using multiple technologies including MALDI-TOF mass spectrometry, dialysis assay and X-ray crystallographic studies etc. This functional group was also successfully applied to discovery of a new JAK3 inhibitor, suggesting its potential application in designing other kinase inhibitors.

Sunday, October 3, 2021

Covalent Inhibition of SARS-CoV-2 RBD-ACE2 Interaction by Aptamers with Multiple Sulfur(VI) Fluoride Exchange Modifications

Qin Z, Zhu Y, Xiang Y. 

ChemRxiv. 2021

The SARS-CoV-2 spike protein uses its receptor-binding domain (RBD) to interact with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells, establishing the first step of SARS-CoV-2 infection. Inhibitors of RBD-ACE2 interaction, therefore, have shown great promise in preventing SARS-CoV-2 infection. Currently known RBD-ACE2 inhibitors are all based on reversible binding and must compete with ACE2 or RBD at the equilibrium. On the other hand, covalent inhibitors, such as those based on sulfur(VI) fluoride exchange (SuFEx) chemistry, can form irreversible chemical bonds with target proteins and offer advantages including higher potency and longer duration of inhibition. Here, we report covalent aptamer inhibitors that can block RBD-ACE2 by forming covalent bonds with RBD. These covalent aptamer inhibitors were developed by equipping known RBD aptamers with multiple SuFEx (mSuFEx) modifications. The mSuFEx-aptamer 6C3-7SF underwent strong covalent bonding with RBD and some of its variants at fast rates (t1/2 = 20 ~ 29 min−1) and induced more efficient RBD-ACE2 inhibition (IC50 = 26 ~ 37 nM) than the original aptamer (IC50 > 200 nM) according to an in vitro enzyme-linked immunosorbent assay (ELISA). The covalent bond formation was highly selective to RBD over human serum albumin (HSA) and ACE2, and could occur efficiently in diluted human serum. Peptide fragmentation analyses of the RBD-6C3-7SF adducts revealed multiple sites of covalent bonding on RBD, including K378, R408, Y422, Y424, Y453, and K458. The surprising R408 suggests that context-specific non-N-terminal arginine could be a new type of targetable residue by SuFEx-based covalent inhibitors, which were never reported as reactive with any non-N-terminal arginine in target proteins. In addition, RBD R408 is responsible for binding with ACE2 N90 glycan, and this arginine is conserved in SARS-CoV-2 variants of concern or interest, suggesting that R408 could be the potential site of interest for developing SuFEx-based covalent inhibitors against threatening SARS-CoV-2 variants. Although the compatibility of mSuFEx-based covalent aptamers in cellular and in vivo systems should be further investigated, our study demonstrated the promise of mSuFEx chemistry in constructing potent covalent aptamers to inhibit important protein-protein interactions (PPIs).

Saturday, October 2, 2021

Covalent inhibitor targets KRasG12C: a new paradigm for drugging the undruggable and challenges ahead

 Hui-yu Li, Wei-liang Qi, Yu-xiang Wang, Ling-hua Meng

 Genes & Diseases, 2021

KRAS is one of the most commonly mutated oncogenes in cancers and therapeutics directly targeting the KRas have been challenging. Among the different known mutants, KRasG12C has been proved to be successfully targeted recently. Several covalent inhibitors selectively targeting KRasG12C have shown promising efficacy against cancers harboring KRASG12C mutation in clinical trials and AMG510 (sotorasib) has been approved for the treatment of KRASG12C-mutated locally advanced or metastatic non-small cell lung cancer. However, the overall responsive rate of KRasG12C inhibitors was around 50% in patients with non-small cell lung cancer and the efficacy in patients with colorectal cancer or appendiceal cancer appears to be less desirable. It is of great importance to discover biomarkers to distinguish patients who are likely benefitted. Moreover, adaptive resistance would occur inevitably with the persistent administration like other molecularly targeted therapies. Several combinatorial regimens have been studied in an effort to potentiate the efficacy of KRasG12C inhibitors in preclinical settings. This review summarized the recent progress of covalent KRasG12C inhibitors with a focus on identifying biomarkers to predict or monitor the efficacy and proposing rational drug combinations based on elucidation of the mechanisms of drug resistance.

Thursday, September 30, 2021

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

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.

Wednesday, September 29, 2021

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

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.

Saturday, September 18, 2021

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).

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.

Friday, September 17, 2021

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

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.

Sunday, September 12, 2021

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

    • 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.

Saturday, September 11, 2021

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 Chemistry2021

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.

Thursday, September 2, 2021

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

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.

Tuesday, August 31, 2021

A sulfonyl fluoride derivative inhibits EGFRL858R/T790M/C797S by covalent modification of the catalytic lysine

Francesca Ferlenghi, Laura Scalvini, Federica Vacondio, Riccardo Castelli, Nicole Bozza, Giuseppe Marseglia, Silvia Rivara, Alessio Lodola, Silvia La Monica, Roberta Minari, Pier Giorgio Petronini, Roberta Alfieri, Marcello Tiseo, Marco Mor,

European Journal of Medicinal Chemistry, 225, 2021, 113786,

The emergence of the C797S mutation in EGFR is a frequent mechanism of resistance to osimertinib in the treatment of non-small cell lung cancer (NSCLC). In the present work, we report the design, synthesis and biochemical characterization of UPR1444 (compound 11), a new sulfonyl fluoride derivative which potently and irreversibly inhibits EGFRL858R/T790M/C797S through the formation of a sulfonamide bond with the catalytic residue Lys745. Enzymatic assays show that compound 11 displayed an inhibitory activity on EGFRWT comparable to that of osimertinib, and it resulted more selective than the sulfonyl fluoride probe XO44, recently reported to inhibit a significant part of the kinome. Neither compound 11 nor XO44 inhibited EGFRdel19/T790M/C797S triple mutant. When tested in Ba/F3 cells expressing EGFRL858R/T790M/C797S, compound 11 resulted significantly more potent than osimertinib at inhibiting both EGFR autophosphorylation and proliferation, even if the inhibition of EGFR autophosphorylation by compound 11 in Ba/F3 cells was not long lasting.

Sunday, August 29, 2021

A Potent Fluorescent Reversible-Covalent Inhibitor of Cardiac Muscle Contraction

 Fangze Cai, Thomas Kampourakis, Brittney A. Klein, and Brian D. Sykes

ACS Medicinal Chemistry Letters 2021
DOI: 10.1021/acsmedchemlett.1c00366

Compounds that directly modulate the response of the cardiac sarcomere have potential in the treatment of cardiac disease. While a number of sarcomere activators have been discovered and extensively studied, very few inhibitors have been identified. We report a potent cardiac sarcomere inhibitor, DN-F01, targeting the cardiac muscle thin filament protein troponin complex. Functional studies show that DN-F01 has a strong inhibitory calcium-dependent effect on cardiac myofibrillar ATPase activity with an IC50 value of 11 ± 4 nmol/L. DN-F01 is shown to bind to a cardiac troponin C–troponin I chimera (cChimera) with a KD of ∼50 nM using fluorescence spectroscopy, indicating that troponin is the likely target for DN-F01. NMR titrations of DN-F01 to C35S and A-Cys cChimera show covalent and noncovalent binding of DN-F01 bound to the calcium-saturated cChimera.

Monday, August 23, 2021

Activity-Based Hydrazine Probes for Protein Profiling of Electrophilic Functionality in Therapeutic Targets

Zongtao Lin, Xie Wang, Katelyn A. Bustin, Kyosuke Shishikura, Nate R. McKnight, Lin He, Radu M. Suciu, Kai Hu, Xian Han, Mina Ahmadi, Erika J. Olson, William H. Parsons, and Megan L. Matthews

ACS Central Science 2021

DOI: 10.1021/acscentsci.1c00616

Most known probes for activity-based protein profiling (ABPP) use electrophilic groups that tag a single type of nucleophilic amino acid to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electrophilic enzyme cofactors, transient intermediates, and labile regulatory modifications, but ABPP probes for such species are underdeveloped. Here, we describe a versatile class of probes for this less charted hemisphere of the proteome. The use of an electron-rich hydrazine as the common chemical modifier enables covalent targeting of multiple, pharmacologically important classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment occurs by both polar and radicaloid mechanisms, can be blocked by molecules that occupy the active sites, and depends on the proper poise of the active site for turnover. These traits will enable the probes to be used to identify specific inhibitors of individual members of these multiple enzyme classes, making them uniquely versatile among known ABPP probes.

Thiol Reactivity of N-Aryl α-Methylene-γ-lactams: Influence of the Guaianolide Structure [@KayBrummond]

 Daniel P. Dempe, Chong-Lei Ji, Peng Liu, and Kay M. Brummond The Journal of Organic Chemistry, 2020 DOI: 10.1021/acs.joc.2c01530 The α-meth...