Current research in chemical biology最新文献

Attachment of ubiquitin and ubiquitin-like modifiers (UbLs) are reversible post-translational modification of proteins that regulate crucial cellular functions, ranging from protein homeostasis to the control of protein-protein interactions. In the cell genome, in addition to the high number of E3 ligases to control ubiquitin/UbL conjugation, a considerable number of deubiquitinases (DUBs) and Ubiquitin-like proteases (ULPs) are also encoded, indicating the essential role of the reversible deubiquitinating proteolytic activity. Most DUBs and ULPs are cysteine proteases, containing a nucleophilic cysteine in the active site that cleaves the isopeptide bond between ubiquitin and target substrate or between ubiquitin units in polymeric chains. Significant progress has been made in recent years regarding the identification of novel types of DUBs and ULPs, as well as in our understanding of their molecular mechanisms. This progress has been partially attributed to the development of specific chemical tools, such as Activity-Based Probes (ABPs), designed for studying DUB cysteine proteases. ABPs mimic enzymatic substrates and, in an enzyme-catalyzed reaction manner, remain covalently attached, resembling an irreversible competitive inhibitor. The structures formed by ABPs in complex with enzymes provide valuable insights into catalytic mechanisms and the interactions between ubiquitin and DUBs/ULPs proteases. In this review we will summarize recent advancements in the use of ABPs to characterize the structures of DUBs/ULPs in complex with ubiquitin/UbLs. Additionally, we will present relevant examples of complex structures of DUBs with specific ABPs ubiquitin-linked chains.

Small-molecule chemical probes are crucial tools to study the function of unexplored proteins in biological systems, thereby directly impacting preclinical target validation. Being one of the largest protein families in humans, protein kinases are currently among the most important and fruitful molecular targets in drug discovery. However, a significant fraction of the human “kinome” is still understudied and growing efforts in the scientific community aim at the development of specific chemical tool compounds for such “dark” kinases. Covalent targeting has proven to be a valid and rational strategy towards high-quality chemical probes enabling superior potencies, high selectivities and sustained target engagement. In the kinase field, the targeting of non-catalytic cysteine residues has been particularly fruitful and there is an increasing interest in addressing other residues, such as lysine or tyrosine. Herein, we discuss the properties and generation of covalent kinase inhibitors, with a special emphasis on electrophilic functional groups that can be used as “warheads”. Moreover, we highlight studies showcasing the development of covalent chemical probes targeting cysteine and lysine residues in an irreversible or reversible-covalent manner.

Phosphatidylinositol-5-phosphate 4-kinase gamma (PI5P4Kγ), which phosphorylates phosphatidylinositol-5-monophosphate (PI(5)P), is a human lipid kinase with intriguing roles in inflammation, T cell activation, autophagy regulation, immunity, heart failure, and several cancers. To provide a high-quality chemical tool that would enable additional characterization of this protein, we designed and evaluated a potent, selective, and cell-active inhibitor of human PI5P4Kγ. We describe the use of the PI5P4Kγ NanoBRET assay to generate structure–activity relationships (SAR), support chemical probe (2) design, and identify a structurally related negative control (4). We have characterized the binding of our chemical probe to PI5P4Kγ using orthogonal assay formats reliant on competition with an ATP-competitive reagent. Based on our results in these assays, additional ATP titration studies, and published co-crystal structures with structurally related compounds, we hypothesize that 2 binds in the ATP active site of PI5P4Kγ. Kinome-wide profiling complemented by further off-target screening confirmed the selectivity of both our chemical probe and negative control. When a breast cancer cell line (MCF-7) was treated with compound 2, increased mTORC1 signaling was observed, demonstrating that efficacious binding of 2 to PI5P4Kγ in cells results in activation of a negative feedback loop also reported in PI5P4Kγ knockout mice.

Small molecule modulators are important tools to study both basic biology and the complex signaling of protein kinases. The cdc2-like kinases (CLK) are a family of four kinases that have garnered recent interest for their involvement in a diverse set of diseases such as neurodegeneration, autoimmunity, and many cancers. Targeted medicinal chemistry around a CLK inhibitor hit identified through screening of a kinase inhibitor set against a large panel of kinases allowed us to identify a potent and selective inhibitor of CLK1, 2, and 4. Here, we present the synthesis, selectivity, and preliminary biological characterization of this compound – SGC-CLK-1 (CAF-170). We further show CLK2 has the highest binding affinity, and high CLK2 expression correlates with a lower IC₅₀ in a screen of multiple cancer cell lines. Finally, we show that SGC-CLK-1 not only reduces serine arginine-rich (SR) protein phosphorylation but also alters SR protein and CLK2 subcellular localization in a reversible way. Therefore, we anticipate that this compound will be a valuable tool for increasing our understanding of CLKs and their targets, SR proteins, at the level of phosphorylation and subcellular localization.

Because translating the growing body of knowledge about human disease into treatments has been slower than expected, the application of machine learning techniques to drug repositioning has become attractive. An effective and comprehensive understanding of the current state of drug repositioning can help researchers to investigate more efficient and accurate algorithms. In this study, we first present the theoretical rationale for drug repositioning analysis. Then, we conduct a comprehensive review on machine learning algorithms for drug discovery, which include (1) traditional machine learning-based models using linear and logistic regression, support vector machines, random forest, and decision tree, (2) network transmission-based models using drug–disease similarity and network similarity-based reasoning, (3) matrix completion and matrix factorization-based methods using matrix completion, logistic matrix factorization, collaborative matrix factorization, and regularized matrix factorization, and (4) deep learning-based methods using deep neural networks, convolutional neural networks, recurrent neural networks, and graph convolutional networks. This is followed by a review of commonly used data sources for drug repositioning, as well as an introduction to particular data sources that can be employed by researchers. To conclude, we discuss the future developments and challenges of drug repositioning methods.

Specificity is a limiting factor when using small-molecule inhibitors to study protein kinase signalling. Since inhibitor-resistant kinase mutants (i.e., drug-resistant alleles) remain active in the presence of inhibitor, they facilitate validation of on-target effects. By combining an inhibitor-resistant kinase mutant with mass spectrometry-based phosphoproteomics, we previously devised a systematic strategy for reliable identification and validation of CSNK2 substrates. In this study, we use the same strategy to evaluate the selectivity of CX-4945, a clinical stage CSNK2 inhibitor, and SGC-CK2-1, a chemical probe selectively targeting CSNK2. Human osteosarcoma (U2OS) cells expressing exogenous wild-type CSNK2A1 (WT) or an inhibitor-resistant triple mutant (TM, V66A/H160D/I174A) were treated with CX-4945 or SGC-CK2-1 prior to analysis using triple SILAC (phospho)proteomics. The minority of phosphosites, 15% at 4 ​h and 5% at 24 ​h, that were significantly downregulated in response to CX-4945 treatment were determined to be CSNK2A1-dependent. By comparison, the majority of phosphosites, >55% at both 4 and 24 ​h, that were significantly downregulated in response to SGC-CK2-1 were identified as CSNK2A1-dependent. This indicates that SGC-CK2-1 exhibits significantly greater selectivity towards CSNK2A1 than CX-4945. Notably, utilization of SGC-CK2-1 in cells expressing CSNK2A1-TM enabled the identification of >300 CSNK2A1-dependent phosphosites. Overall, this study highlights the utility of exploiting highly selective chemical probes together with inhibitor-resistant kinase mutants to facilitate identification of bona fide kinase substrates.

Protein ubiquitination is involved in almost all aspects of eukaryotic biology. To investigate the cellular role of ubiquitin (Ub) system, a variety of Ub-based tools have been developed. In comparison to these laborious chemical synthesis, enzymatic methods offer good alternatives for the production of the desired Ub tools from the recombinant protein building blocks and are therefore easier to implement in the typical biological research laboratories. In this minireview, we summarize some key enzymatic approaches for the preparation of Ub-based tools.

Ferulic acid decarboxylase (Fdc) is a member of the microbial UbiD superfamily, a diverse family of (de)carboxylases capable of reversible decarboxylation on α,β-unsaturated acids. Recent application of Fdc includes in vivo generation of hydrocarbons such as isobutene and 1,3-butadiene, as well as C–H activation through CO₂ fixation. Protein engineering has expanded the substrate scope of the Aspergillus niger ferulic acid decarboxylase (AnFdc) to include (hetero)aromatic acid substrates. To further improve activity with aromatic acids, we introduced disulphide bonds into AnFdc to generate more thermostable variants. While some variants are negatively affected in co-factor incorporation and thus activity, others display increased thermostability and enhanced activity. The most thermostable disulphide bond AnFdc variant was combined with key active site mutations, allowing access to improved (hetero)aromatic decarboxylation including naphthoic acid decarboxylation. The reverse process, naphthalene carboxylation, is relevant to understanding microbial UbiD-mediated anaerobic naphthalene/benzene degradation. The improved naphthoic acid decarboxylation achieved here suggests further scope for AnFdc evolution towards an amenable model system for aromatic C–H activation through carboxylation.

Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT) and animal trypanosomiases, cycles between a bloodstream form in mammals and a procyclic form in the gut of its insect vector. We previously discovered that the human bromodomain inhibitor I-BET151 causes transcriptome changes that resemble the transition from the bloodstream to the procyclic form. In particular, I-BET151 induces replacement of variant surface glycoprotein (VSG) with procyclin protein. While modest binding of I-BET151 to TbBdf2 and TbBdf3 has been demonstrated, it is unknown whether I-BET151 binds to other identified T. brucei bromodomain proteins and/or other targets. To identify target(s) in T. brucei, we have synthesized I-BET151 derivatives maintaining the key pharmacophoric elements with functionality useful for chemoproteomic approaches. We identified compounds that are potent in inducing expression of procyclin, delineating a strategy towards the design of drugs against HAT and other trypanosomiases. Furthermore, these derivatives represent useful chemical probes to elucidate the molecular mechanism underlying I-BET151-induced differentiation.

The rapid spread of COVID-19 has caused a worldwide public health crisis. For prompt and effective development of antivirals for SARS-CoV-2, the pathogen of COVID-19, drug repurposing has been broadly conducted by targeting the main protease (M^Pro), a key enzyme responsible for the replication of virus inside the host. In this study, we evaluate the inhibition potency of a nitrothiazole-containing drug, halicin, and reveal its reaction and interaction mechanism with M^Pro. The in vitro potency test shows that halicin inhibits the activity of M^Pro an IC₅₀ of 181.7 ​nM. Native mass spectrometry and X-ray crystallography studies clearly indicate that the nitrothiazole fragment of halicin covalently binds to the catalytic cysteine C145 of M^Pro. Interaction and conformational changes inside the active site of M^Pro suggest a favorable nucleophilic aromatic substitution reaction mechanism between M^Pro C145 and halicin, explaining the high inhibition potency of halicin towards M^Pro.