ACS Chemical Biology最新文献

Inositol-requiring enzyme 1α (IRE1α) is a sensor of endoplasmic reticulum (ER) stress and drives ER stress response pathways. Activated IRE1α exhibits RNase activity and cleaves mRNA encoding X-box binding protein 1, a transcription factor that induces the expression of genes that maintain ER proteostasis for cell survival. Previously, we showed that IRE1α undergoes S-nitrosylation, a post-translational modification induced by nitric oxide (NO), resulting in reduced RNase activity. Therefore, S-nitrosylation of IRE1α compromises the response to ER stress, making cells more vulnerable. We conducted virtual screening and cell-based validation experiments to identify compounds that inhibit the S-nitrosylation of IRE1α by targeting nitrosylated cysteine residues. We ultimately identified a compound (1ACTA) that selectively inhibits the S-nitrosylation of IRE1α and prevents the NO-induced reduction of RNase activity. Furthermore, 1ACTA reduces the rate of NO-induced cell death. Our research identified S-nitrosylation as a novel target for drug development for IRE1α and provides a suitable screening strategy.

Pleckstrin homology (PH) domains are structural motifs critical for cellular processes, such as signal transduction and cytoskeletal organization. Due to their involvement in various diseases, PH domains are promising therapeutic targets, yet their highly charged and hydrophobic binding sites are not ideal for traditional small drugs. In this study, we designed a DNA-encoded library (DEL) mimicking phospholipids to identify novel modulators targeting PH domains with uncharted chemical properties. Screening against several PH domains led to the discovery of 2DII, a small molecule that selectively binds to mSin1^PH. This compound can modulate mTORC2 activity by impairing mTORC2's membrane interactions, resulting in reduced AKT1 phosphorylation. A micromapping via Dexter energy transfer based on 2DII bearing an iridium catalyst (2DII-Ir), along with a biotin-diazirine small molecule was used for target identification by proteomics, which confirmed mSin1 as the primary intracellular target of 2DII, demonstrating its potential for selective mTORC2 pathway modulation. These findings introduce a novel strategy for targeting PH domains and provide a foundation for the development of therapeutic interventions that modulate PH-domain-dependent signaling pathways.

The use of photoresponsive ligands to optically control proteins of interest, known as photopharmacology, is a powerful technique for elucidating cellular function in living cells and animals with a high spatiotemporal resolution. The adenosine A_2A receptor (A_2AR) is a G protein-coupled receptor that is expressed in various tissues; its dysregulation is implicated in severe diseases such as insomnia and Parkinson's disease. A detailed elucidation of the physiological function of A_2AR is, therefore, highly desirable. In the present study, we developed two photoswitchable ligands, photoAd(blue) and photoAd(vio), that target A_2AR. Using photoAd(vio), we successfully demonstrated the selective activation of A_2AR in living cells by violet-light irradiation with high spatiotemporal resolution.

Nonribosomal peptide synthetases (NRPSs) produce diverse natural products including siderophores, chelating agents that many pathogenic bacteria produce to survive in low iron conditions. Engineering NRPSs to produce diverse siderophore analogs could lead to the generation of novel antibiotics and imaging agents that take advantage of this unique iron uptake system in bacteria. The highly pathogenic and antibiotic-resistant bacteria Acinetobacter baumannii produces fimsbactin, an unusual branched siderophore with iron-binding catechol groups bound to a serine or threonine side chain. To explore the substrate promiscuity of the assembly line enzymes, we report a structure-guided investigation of the stand-alone aryl adenylation enzyme FbsH. We report structures bound to its native substrate 2,3-dihydroxybenzoic acid (DHB) as well as an inhibitor that mimics the adenylate intermediate. We produced enzyme variants with an expanded binding pocket that are more tolerant for analogs containing a DHB C4 modification. Wild-type and mutant enzymes were then used in an in vitro reconstitution analysis to assess the production of analogs of the final product as well as several early stage intermediates. This analysis shows that some altered substrates progress down the fimsbactin assembly line to the downstream domains. However, analogs from alternate building blocks are produced at lower levels, indicating that selectivity exists in the downstream catalytic domains. These findings expand the substrate scope of producing condensation products between serine and aryl acids and identify the bottlenecks for chemoenzymatic production of fimsbactin analogs.

The E3 ligase HOIP is vital for the NF-κB pathway and is implicated in cancer and immunity. However, it remains challenging to achieve high selectivity by directly targeting the conserved catalytic RBR domain of HOIP. Herein, we identified four low-molecular-weight compounds that bind to an uncharacterized pocket of the HOIP PUB domain (HOIP^PUB). The complex structure facilitated the discovery of the first single-digit micromolar ligand of HOIP^PUB, tolfenamic acid, which exhibited over 30-fold selectivity due to the low sequence identity of the uncharacterized pocket of HOIP^PUB. Although tolfenamic acid did not block the substrate recognition and linear ubiquitination activity of HOIP, a ligand of the uncharacterized PUB pocket of HOIP (LUPH), by chemical linking pomalidomide with tolfenamic acid, degraded HOIP, reduced NEMO ubiquitination and p65 phosphorylation, and eventually inhibited NF-κB activation and breast cancer cell proliferation. Our work proposes an alternative strategy to target the nonfunctional pocket of the PUB domain with high sequence diversity to promote HOIP degradation, rather than targeting the conserved RBR domain to block the catalytic function of HOIP.

Precise cell elimination within intricate cellular populations is hampered by issues arising from the multifaceted biological properties of cells and the expansive reactivity of chemical agents. Current chemical platforms are often limited by their complexity, toxicity, and poor physical/chemical properties. Here, we report on the synthesis of a structurally versatile library of chemically tunable bisacylphosphane oxides (BAPOs), which harnesses the spatiotemporal precision of light delivery, thereby establishing a universal strategy for on-demand, precise cellular ablation in vitro and in vivo.

Many complex terpenoids, predominantly isolated from plants and fungi, show drug-like physicochemical properties. Recent advances in genome mining revealed actinobacteria as an almost untouched treasure trove of terpene biosynthetic gene clusters (BGCs). In this study, we characterized a terpene BGC with an unusual architecture. The selected BGC includes, among others, genes encoding a terpene cyclase fused to a truncated reductase domain and a cytochrome P450 monooxygenase (P450) that is split over three gene fragments. Functional characterization of the BGC in a heterologous host led to the identification of several new members of the trans-eunicellane family of diterpenoids, the euthailols, that feature unique oxidation patterns. A combination of bioinformatic analyses, structural modeling studies, and heterologous expression revealed a dual function of the pathway-encoded hypothetical protein that acts as an isomerase and an oxygenase. Moreover, in the absence of other tailoring enzymes, a P450 hydroxylates the eunicellane scaffold at a position that is not modified in other eunicellanes. Surprisingly, both the modifications installed by the hypothetical protein and one of the P450s exhibit partial redundancy. Bioactivity assays revealed that some of the euthailols show growth inhibitory properties against Gram-negative ESKAPE pathogens. The characterization of the euthailol BGC in this study provides unprecedented insights into the partial functional redundancy of tailoring enzymes in complex diterpenoid biosynthesis and highlights hypothetical proteins as an important and largely overlooked family of tailoring enzymes involved in the maturation of complex terpenoids.

Protein mycoloylation is a recently identified unusual post-translational modification (PTM) exclusively observed in Mycobacteriales, an order of bacteria that includes several human pathogens. These bacteria possess a distinctive outer membrane, known as the mycomembrane, composed of very long-chain fatty acids called mycolic acids. It has been demonstrated that a few mycomembrane proteins undergo covalent modification with mycolic acids in the model organism Corynebacterium glutamicum through the action of mycoloyltransferase MytC. This PTM represents the first example of protein O-acylation in prokaryotes and also the first example of protein modification by mycolic acid. Many questions about the specificity of protein O-mycoloylation remain crucial for understanding its evolutionary significance in Mycobacteriales and its role in cell physiology. We have developed the first bioorthogonal mycolate donor featuring the natural mycolic acid pattern, enabling direct, unambiguous transfer of the lipid moiety to its acceptors and efficient metabolic labeling and enrichment of MytC protein substrates. Mass spectrometry analysis of the labeled proteins and comparative proteomic analysis of the cell envelope proteome between wild-type and ΔmytC strains identified an unbiased list of 21 proteins likely mycoloylated in the cell. The robustness of our approach is demonstrated by the successful biological validation of mycoloylation in 6 candidate proteins within wild-type cells, revealing the characteristic profile of proteins modified with natural mycolates. These findings provide interesting insights into the significance of this new lipidation pathway and pave the way for understanding their function, especially concerning the mycoloyltransferase family that includes the essential Antigen85 enzymes in Mycobacteria.

Pleurotin is a meroterpenoid specialized metabolite made by the fungus Hohenbuehelia grisea, and it is a lead anticancer molecule due to its irreversible inhibition of the thioredoxin-thioredoxin reductase system. Total synthesis of pleurotin has been achieved, including through a stereoselective route; however, its biosynthesis has not been characterized. In this study, we used isotope-labeled precursor feeding to show that the nonterpenoid quinone ring of pleurotin and its congeners is derived from phenylalanine. We sequenced the genome of H. grisea and used comparative transcriptomics to identify putative genes involved in pleurotin biosynthesis. We heterologously expressed a UbiA-like prenyltransferase from H. grisea that led to the accumulation of the first predicted pleurotin biosynthetic intermediate, 3-farnesyl-4-hydroxybenzoic acid. This work sets the foundation to fully elucidate the biosynthesis of pleurotin and its congeners, with long-term potential to optimize their production for therapeutic use and engineer the pathway toward the biosynthesis of valuable analogues.

Gemcitabine-based monotherapy or combination therapy has become the standard treatment for locally advanced and metastatic pancreatic cancer. However, the emergence of resistance within weeks of treatment severely compromises therapeutic efficacy. The intricate biological process of gemcitabine resistance in pancreatic cancer presents a complex challenge, as the underlying mechanisms remain unclear. Identifying the target protein of gemcitabine is crucial for studying its drug-resistance mechanism. An activity-based probe is a powerful tool for studying drug target proteins, but the current lack of activity-based gemcitabine probes with robust biological activity hinders research on gemcitabine. In this study, we developed three active probes based on gemcitabine, among which Gem-3 demonstrated excellent stability and labeling efficacy. We utilized Gem-3 in conjunction with chemical proteomics to identify intracellular target proteins. We identified 79 proteins that interact with gemcitabine, most of which were previously unknown and represented various functional classes. Additionally, we validated the increased expression of IFIT3 and MARCKS in drug-resistant cells, along with the activation of the NF-κB signaling pathway. These findings substantially contribute to our comprehension of gemcitabine’s target proteins and further our understanding of the mechanisms driving gemcitabine resistance in pancreatic cancer cells.