ACS Central Science最新文献

Cyclooxygenase-2 (COX-2) is an enzyme that plays a pivotal role in peripheral inflammation and pain via the prostaglandin pathway. In the central nervous system (CNS), COX-2 is implicated in neurodegenerative and psychiatric disorders as a potential therapeutic target and biomarker. However, clinical studies with COX-2 have yielded inconsistent results, partly due to limited mechanistic understanding of how COX-2 activity relates to CNS pathology. Therefore, developing COX-2 positron emission tomography (PET) radiotracers for human neuroimaging is of interest. This study introduces [¹¹C]BRD1158, which is a potent and uniquely fast-binding, selective COX-2 PET radiotracer. [¹¹C]BRD1158 was developed by prioritizing potency at COX-2, isoform selectivity over COX-1, fast binding kinetics, and free fraction in the brain. Evaluated through in vivo PET neuroimaging in rodent models with human COX-2 overexpression, [¹¹C]BRD1158 demonstrated high brain uptake, fast target-engagement, functional reversibility, and excellent specific binding, which is advantageous for human imaging applications. Lastly, post-mortem samples from Huntington’s disease (HD) patients and preclinical HD mouse models showed that COX-2 levels were elevated specifically in disease-affected brain regions, primarily from increased expression in microglia. These findings indicate that COX-2 holds promise as a novel clinical marker of HD onset and progression, one of many potential applications of [¹¹C]BRD1158 human PET.

A COX-2 specific radiotracer for positron emission tomography imaging, [¹¹C]BRD1158, was developed and evaluated in rodent models. [¹¹C]BRD1158 demonstrated rapid binding and functional reversibility and shows promise for human translation. Additionally, evidence for the involvement of COX-2 in Huntington’s disease is presented, highlighting the relevance of [¹¹C]BRD1158 to clinical and research applications in neurodegeneration.

Antiviral nucleoside analogues (e.g., Molnupiravir, Remdesivir) played key roles in the treatment of COVID-19 by targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The nucleoside of Molnupiravir, N⁴-hydroxycytidine (NHC), exists in two tautomeric forms that pair either with G or A within the RdRp active site, causing an accumulation of viral RNA mutations during replication. Detailed insights into the tautomeric states within base pairs and the structural influence of NHC in RNA are still missing. In this study, we investigate the properties of NHC:G and NHC:A base pairs in a self-complementary RNA duplex by UV thermal melting and NMR spectroscopy using atom-specifically ¹⁵N-labeled versions of NHC that were incorporated into oligonucleotides by solid-phase synthesis. NMR analysis revealed that NHC forms a Watson–Crick base pair with G via its amino form, whereas two equally populated conformations were detected for the NHC:A base pair: a weakly hydrogen-bonded Watson–Crick base pair with NHC in the imino form and another conformation with A shifted toward the minor groove. Moreover, we found a variable influence of NHC:G and NHC:A base pairs on the neighboring duplex environment. This study provides conclusive experimental evidence for the existence of two tautomeric forms of NHC within RNA base pairs.

NMR of RNA duplexes containing atom-specifically ¹⁵N-labeled N⁴-hydroxycytidine (NHC) enables us to determine the tautomeric form and the base-pairing pattern of NHC:G and NHC:A.

Glycosylation plays a pivotal role in the intricate landscape of human cholangiocarcinoma (CCA), actively participating in key pathophysiological processes driving tumor progression. Among the various glycosylation modifications, O-linked β-N-acetyl-glucosamine modification (O-GlcNAcylation) emerges as a dynamic regulator influencing diverse tumor-associated biological activities. In this study, we employed a state-of-the-art chemical proteomic approach to analyze intact glycopeptides, unveiling the critical role of O-GlcNAcylation in orchestrating Keratin 18 (K18) and its interplay with tricarboxylic acid (TCA) cycle enzymes, specifically isocitrate dehydrogenases (IDHs), to propel CCA progression. Our findings shed light on the mechanistic intricacies of O-GlcNAcylation, revealing that site-specific modification of K18 on Ser 30 serves as a stabilizing factor, amplifying the expression of cell cycle checkpoints. This molecular event intricately fosters cell cycle progression and augments cellular growth in CCA. Notably, the interaction between O-GlcNAcylated K18 and IDHs orchestrates metabolic reprogramming by down-regulating citrate and isocitrate levels while elevating α-ketoglutarate (α-KG). These metabolic shifts further contribute to the overall tumorigenic potential of CCA. Our study thus expands the current understanding of protein O-GlcNAcylation and introduces a new layer of complexity to post-translational control over metabolism and tumorigenesis.

Synopsis: Unraveling the role of O-GlcNAcylation in cholangiocarcinoma (CCA), our study illuminates how it influences Keratin 18, impacting metabolic reprogramming and CCA progression. These findings deepen our understanding of tumorigenesis and post-translational control over metabolism.

The programmed synthesis of sequence-defined biomaterials whose monomer backbones diverge from those of canonical α-amino acids represents the next frontier in protein and biomaterial evolution. Such next-generation molecules provide otherwise nonexistent opportunities to develop improved biologic therapies, bioremediation tools, and biodegradable plastic-like materials. One monomer family of particular interest for biomaterials includes β-hydroxy acids. Many natural products contain isolated β-hydroxy acid monomers, and polymers of β-hydroxy acids (β-esters) are found in polyhydroxyalkanoate (PHA) polyesters under development as bioplastics and drug encapsulation/delivery systems. Here we report that β²-hydroxy acids possessing both (R) and (S) absolute configuration are substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro and that (S)-β²-hydroxy acids are substrates in cellulo. Using the orthogonal MaPylRS/MatRNA^Pyl synthetase/tRNA pair, in conjunction with wild-type E. coli ribosomes and EF-Tu, we report the cellular synthesis of model proteins containing two (S)-β²-hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed preference for the (S)-β²-hydroxy acid and provide mechanistic insights that inform future engineering efforts. As far as we know, this finding represents the first example of an orthogonal synthetase that acylates tRNA with a β²-hydroxy acid substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers produced in cellulo.

The aminoacyl-tRNA synthetase PylRS from M. alvus acylates tRNA^Pyl with β²-hydroxy acid substrates and supports their incorporation at multiple sites into a protein in vivo.

Current approaches to evaluate molecular complexity use algorithmic complexity, rooted in computer science, and thus are not experimentally measurable. Directly evaluating molecular complexity could be used to study directed vs undirected processes in the creation of molecules, with potential applications in drug discovery, the origin of life, and artificial life. Assembly theory has been developed to quantify the complexity of a molecule by finding the shortest path to construct the molecule from building blocks, revealing its molecular assembly index (MA). In this study, we present an approach to rapidly infer the MA of molecules from spectroscopic measurements. We demonstrate that the MA can be experimentally measured by using three independent techniques: nuclear magnetic resonance (NMR), tandem mass spectrometry (MS/MS), and infrared spectroscopy (IR). By identifying and analyzing the number of absorbances in IR spectra, carbon resonances in NMR, or molecular fragments in tandem MS, the MA of an unknown molecule can be reliably estimated. This represents the first experimentally quantifiable approach to determining molecular assembly. This paves the way to use experimental techniques to explore the evolution of complex molecules as well as a unique marker of where an evolutionary process has been operating.

This study introduces a novel approach to experimentally measure molecular complexity (MA) using nuclear magnetic resonance, tandem mass spectrometry, and infrared spectroscopy.

Thiol-mediated uptake (TMU) is an intriguing enigma in current chemistry and biology. While the appearance of cell-penetrating activity upon attachment of cascade exchangers (CAXs) has been observed by many and is increasingly being used in practice, the molecular basis of TMU is essentially unknown. The objective of this study was to develop a general protocol to decode the dynamic covalent networks that presumably account for TMU. Uptake inhibition patterns obtained from the removal of exchange partners by either protein knockdown or alternative inhibitors are aligned with original patterns generated by CAX transporters and inhibitors and patterns from alternative functions (here cell motility). These inclusive TMU patterns reveal that the four most significant CAXs known today enter cells along three almost orthogonal pathways. Epidithiodiketopiperazines (ETP) exchange preferably with integrins and protein disulfide isomerases (PDIs), benzopolysulfanes (BPS) with different PDIs, presumably PDIA3, and asparagusic acid (AspA), and antisense oligonucleotide phosphorothioates (OPS) exchange with the transferrin receptor and can be activated by the removal of PDIs with their respective inhibitors. These findings provide a solid basis to understand and use TMU to enable and prevent entry into cells.

Methods are developed to show that different, almost orthogonal dynamic covalent networks operate in thiol-mediated uptake to deliver substrates of interest into cells and to reveal which ones are involved.

Chemical control over proton-coupled electron transport via a mediator species drives an artificial electron transport chain between macrocycles.

Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new compound class involving modifications installed by a cytochrome P450, a multinuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-l-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization demonstrated that the P450 enzyme catalyzed the formation of a biaryl C–C cross-link between two Tyr residues with the B12-rSAM generating β-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid, while the methyltransferase acted on the β-carbon of this α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configuration of the atropisomer formed upon biaryl cross-linking. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to isolate new macrocyclic RiPPs biosynthesized via previously undiscovered enzyme chemistry.

Three metalloenzymes and a methyltransferase convert a ribosomally translated peptide into an atropisomeric biaryl macrocyclic peptide with a C-terminal β-amino-α-keto acid and two C-methylations.

Electron transport chains (ETCs) are ubiquitous in nearly all living systems. Replicating the complexity and control inherent in these multicomponent systems using ensembles of small molecules opens up promising avenues for molecular therapeutics, catalyst design, and the development of innovative energy conversion and storage systems. Here, we present a noncovalent, multistep artificial electron transport chains comprising cyclo[8]pyrrole (1), a meso-aryl hexaphyrin(1.0.1.0.1.0) (naphthorosarin 2), and the small molecules I₂ and trifluoroacetic acid (TFA). Specifically, we show that 1) electron transfer occurs from 1 to give I₃^– upon the addition of I₂, 2) proton-coupled electron transfer (PCET) from 1 to give H₃2^•2+ and H₃2⁺ upon the addition of TFA to a dichloromethane mixture of 1 and 2, and 3) that further, stepwise treatment of 1 and 2 with I₂ and TFA promotes electron transport from 1 to give first I₃^– and then H₃2^•2+ and H₃2⁺. The present findings are substantiated through UV-vis-NIR, ¹H NMR, electron paramagnetic resonance (EPR) spectroscopic analyses, cyclic voltammetry studies, and DFT calculations. Single-crystal structure analyses were used to characterize compounds in varying redox states.

Presented is an example of a proton-coupled electron transfer (PCET) system comprising an artificial electron transport chain (ETC) that can be regulated by small molecules or concentration control.