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.

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...