ChemBioChem最新文献

Panepoxydone is a natural NF-κB inhibitor isolated from basidiomycetes belonging to the genus Panus and Lentinus. It is biosynthesized from prenylhydroquinone through successive hydroxylation, epoxidation, and reduction reactions. In this study, we establish an efficient precursor-directed biosynthesis strategy for the structural expansion of panepoxydone based on its biosynthetic pathway. Supplementation of the panepoxydone-producing strain, Panus rudis, with various prenylhydroquinone analogues enabled the production of fourteen previously undescribed panepoxydone derivatives, panepoxyquinoid A-N (2-14). The obtained panepoxydone derivatives together with their parental molecules were evaluated for their inhibitory activity on LPS-induced NO production in RAW 264.7 cells. Compounds 1, 5-6, 10-11, and 14-15 displayed significant suppressive effects on LPS-induced NO production with IC₅₀ values ranging from 4.3 to 30.1 μM.

We have examined in this contribution the electrostatic interactions between single arginine and aspartic acid by analyzing the peptide-peptide binding characteristics involving arginine-aspartic acid, arginine-glycine, arginine-tryptophan and tryptophan-glycine interactions. The results of aspartic acid mutagenesis revealed that the interactions between arginine and aspartic acid have significant dependence on the position and composition of amino acids. While the primary interaction can be attributed to arginine-tryptophan contacts originated from the indole moieties with the main chains of 14-mers containing N-H and C=O moieties, pronounced enhancement could be identified in association with the electrostatic side-chain-side-chain interactions between arginine and aspartic acid. An optimal separation of 2~4 amino acids between two adjacent aspartic acid and tryptophan binding sites can be identified to achieve maximal enhancement of binding interactions. Such observed separation dependence may be utilized to unravel cooperative effects in heterogeneous interactions between single pair of amino acids.

Identifying target proteins that interact with bioactive molecules is indispensable for understanding their mechanisms of action. In this study, we developed a uniform ribosome display technology using equal-length DNAs and mRNAs to improve molecular display principle for target identification. The equal-length DNAs were designed to contain various coding sequences for full-length proteins with molecular weights of up to 130 kDa and were used to synthesize equal-length mRNAs, which allowed the formation of full-length protein-ribosome-equal-length mRNA complexes. Uniform ribosome display selections of dihydrofolate reductase and haloalkane dehalogenase mutant were performed against methotrexate and chlorohexane, respectively. Quantitative changes of proteins after each selection indicated that the target protein-displaying ribosomal complexes were specifically selected through non-covalent or covalent interactions with the corresponding bioactive molecules. Furthermore, selection of full-length proteins interacting with methotrexate or anti-DDX46 antibody from protein pools showed that only the target proteins could be precisely identified even though the molar amounts of equal-length mRNAs encoding them were adjusted to 1/20,000 of the total equal-length mRNAs. Thus, the uniform ribosome display technology enabled efficient identification of target proteins that interact with bioactive small and large molecules through simplified operations without deep sequencing.

Hydrolysis-resistant RNA-peptide conjugates that mimic peptidyl-tRNAs are often required for structural and functional studies of protein synthesis at the ribosome. These conjugates can be synthesized by solid-phase chemical synthesis, which allows maximum flexibility in both the peptide and RNA sequence. The commonly used strategy is based on (3'-N-aminoacyl)-3'-amino-3'-deoxyadenosine solid supports, which already contain the first C-terminal amino acid of the target peptidyl chain. This is a limitation in the sense that different individual supports must be synthesized for different C-terminal amino acids. In this study, we demonstrate a solution to this problem by introducing a novel universal support. The key is a free ribose 3'-NH₂ group that can be coupled to any amino acid. This is made possible by a photocleavable ether moiety that links the ribose 2'-O to the support, thus avoiding the typical O-to-N migration that occurs when using 2'-O-acyl linked solid supports. Once assembled, the conjugate is readily cleaved by UV irradiation. The structural integrity of the obtained peptidyl-RNA conjugates was verified by mass spectrometry analysis. In conclusion, the new photocleavable solid support makes the synthesis of 3'-peptidyl tRNA mimics of different peptidyl chains significantly more efficient compared to the commonly used approaches.

Rubrobacter radiotolerans nerolidol synthase (NerS) and trans-α-bergamotene synthase (BerS) are among the first terpene synthases (TPSs) discovered from thermotolerant bacteria, and, despite sharing the same substrate, make terpenoid products with different carbon scaffolds. Here, the potential thermostability of NerS and BerS was investigated, and NerS was found to retain activity up to 55 °C. A library of 22 NerS and BerS variants was designed to probe the differing reaction mechanisms of NerS and BerS, including residues putatively involved in substrate sequestration, cation-π stabilisation of reactive intermediates, and shaping of the active site contour. Two BerS variants showed improved in vivo titres vs the WT enzyme, and also yielded different ratios of the related sesquiterpenoids (E)-β-farnesene and trans-α-bergamotene. BerS-L86F was proposed to encourage substrate isomerisation by cation-π stabilisation of the first cationic intermediate, resulting in a greater proportion of trans-α-bergamotene. By contrast, BerS-S82L significantly preferred (E)-β-farnesene formation, attributed to steric blocking of the isomerisation step, consistent with what has been observed in several plant TPSs. Our work highlights the importance of isomerisation as a key determinant of product outcome in TPSs, and shows how a combined computational and experimental approach can characterise TPSs and variants with improved and altered functionality.

Cancer has long been a significant threat to human life and health. The advent of immune checkpoint blockade strategies has reversed cancer-induced immune suppression, advanced the development of immunotherapy, and offered new hope in the fight against cancer. Aptamers, which possess the same specificity and affinity as antibodies, are advantageous due to their synthetic accessibility and ease of modification, providing novel insights for immune checkpoint research. In this review, we outline the key aptamers currently developed for immune checkpoints such as CTLA-4, PD-1, PD-L1 and Siglec-15. We explore their potential in therapeutic strategies, including functionalizing or engineering aptamers for covalent binding, valency control, and nanostructure assembly, as well as investigating molecular mechanisms such as glycosylated protein functions and cell-cell interactions. Finally, the future applications of aptamers in immunotherapy are discussed.

Hepatocellular carcinoma (HCC) is recognized globally as one of the most lethal tumors, presenting a significant menace to patients' lives owing to its exceptional aggressiveness and tendency to recur. Transcatheter hepatic arterial chemoembolization (TACE) therapy, as a first-line treatment option for patients with advanced HCC, has been proven effective. However, it is disheartening that nearly 40 % of patients exhibit resistance to this therapy. Consequently, this review delves into the metabolic aspects of glucose metabolism to explore the underlying mechanisms behind TACE treatment resistance and to propose potentially fruitful therapeutic strategies. The ultimate objective is to present novel insights for the development of personalized treatment methods targeting HCC.

The severe acute respiratory syndrome virus 2 (SARS-CoV-2) seriously impacted public health. The evolutionarily conserved viral chymotrypsin-like main protease (M^pro) is an important target for anti-SARS-CoV-2 drug development. Previous studies have shown that the eight N-terminal amino acids (N8) of SARS-CoV M^pro are essential for its dimerization, and are used to design inhibitors against SARS-CoV M^pro dimerization. Here, we established a simple readout assay using SDS-PAGE and Coomassie blue staining to measure inhibitory activity of N8 peptide derived from SARS-CoV-2 M^pro. To optimize its inhibitory effect, we then modified the side-chain length, charge, and hydrophilicity of the N8 peptide, and introduced a mutated M^pro recognition sequence. As a result, we obtained a series of potent peptide inhibitors against SARS-CoV-2 M^pro, with N8-A24 being the most efficient with an IC₅₀ value of 1.44 mM. We observed that N8-A24 reduced M^pro dimerization with an IC₅₀ value of 0.86 mM. Molecular docking revealed that N8-A24 formed hydrogen bond interactions with critical dimeric interface residues, thus inhibiting its dimerization and activity. In conclusion, our study not only discovers a series of peptide inhibitors targeting the SARS-CoV-2 M^pro dimerization, but also provides a promising strategy for the rational design of new inhibitors against COVID-19.

Protein acetylation and acylation are widespread post-translational modifications (PTMs) in eukaryotic and prokaryotic organisms. Histone acetyltransferase (HATs) enzymes catalyze the addition of short-chain acyl moieties to lysine residues on cellular proteins. Many HAT members are found to be dysregulated in human diseases, especially oncological processes. Screening potent and selective HAT inhibitors has promising application for therapeutic innovation. A biochemical assay for quantification of HAT activity utilizing luminescent output is highly desirable to improve upon limitations associated with the classic radiometric assay formats. Here we report the design of a bioluminescent technological platform for robust and sensitive quantification of HAT activity. This platform utilizes the metabolic enzyme acetyl-CoA synthetase 1 (ACS1) for a coupled reaction with firefly luciferase to generate luminescent signal relative to the HAT-catalyzed acetylation reaction. The biochemical assay was implemented in microtiter plate format and our results showed this assay sensitively detected catalytic activity of HAT enzyme p300, accurately measured its steady-state kinetic parameters of histone acetylation and measured the inhibitory potency of HAT inhibitor. This platform demonstrated excellent robustness, reproducibility, and signal-to-background ratios, with a screening window Z'=0.79. Our new bioluminescent design provides an alternative means for HAT enzymatic activity quantitation and HAT inhibitor screening.

Engineering of nonribosomal peptide synthetases (NRPSs) could transform the production of bioactive natural product derivatives. A number of recent reports have described the engineering of NRPSs without marked reductions in yield. Comparative analysis of evolutionarily related NRPSs can provide insights regarding permissive fusion sites for engineering where recombination may occur during evolutionary processes. Studies involving engineering of NRPSs using these recombination sites showed that they have great potential. Moreover, we highlight recent advances in engineering of NRPSs using CRISPR-associated protein 9 (Cas9)-based gene editing technology. The use of Cas9 facilitates the editing of even larger biosynthetic gene clusters (BGCs) close to or over 100 kb in size by precisely targeting and digesting DNA sequences at specific sites. This technology combined with growing understanding of potential fusion sites from large-scale informatics analyses will accelerate the scalable exploration of engineered NRPS assembly lines to obtain bioactive natural product derivatives in high yields.