ACS Bio & Med Chem Au最新文献

Surface Plasmon Resonance (SPR) has proven to be one of the most effective technologies in terms of specificity, affinity, and determination of kinetic parameters for evaluating interactions between macromolecules. The focus of this tutorial is to give an overview of the recent advances and applications of SPR biosensors in biomedicine that are presented emphasizing the potentiality for the detection of very low abundant compounds, which, in recent years, have assumed great importance for prevention and early diagnosis of various diseases in biomedicine. The real-time detection of important biomarkers such as tumor markers, viruses, and toxins but also of compounds of interest such as drugs and hormones allows point-of-care analysis and monitoring of disease progression quickly and in a less invasive manner. Over the past years, several technical innovations have been introduced to SPR devices, which have gone through a process of miniaturization, portability, flexibility, and cost reduction. These characteristics are in line with the advantages of SPR biosensors over other biosensing techniques, i.e., to be label-free detection systems and their capacity to observe in real-time the interactions between a variety of molecules of interest at the metal surface. Recent advances in SPR sensor technology, such as LSPR, SPRi, and SPRM, attempted to improve the sensitivity and performance of molecule detection.

The biological role of DNA and RNA G-quadruplexes (G4s) is relayed to the cellular circuitry by a plethora of proteins known as G4-binding proteins (G4PBs). It is thus critical to decipher the formation of the G4/G4BP complexes to accurately understand their involvement in G4-mediated regulatory mechanisms. While hundreds of G4-interacting compounds (G4 ligands) have been used to uncover G4 biology in a rather indirect manner, only a handful of multivalent ligands allowing for identifying associated proteins have been designed, mostly relying on the covalent capture of G4BPs upon photoactivation. We report herein on such a multivalent ligand named photoMultiTASQ, which belongs to a family of biomimetic and smart G4 ligands known as template-assembled synthetic G-quartets (TASQs). We show how photoMultiTASQ interacts with and photo-cross-links both DNA/RNA G4s and related G4BPs, and develop a new mass spectrometry (MS)-based technique to characterize the covalent adducts. Collectively, these results make photoMultiTASQ a new molecular tool in the arsenal of chemical cross-linking and isolation by pull-down (Chem-CLIP) technologies that uniquely identify targets and validate target engagement in cells.

Genome editing tools have great potential for medicinal use. Among them, zinc finger nucleases (ZFNs) are smaller in size than transcriptional activator-like effector nucleases and CRISPR-Cas9. Therefore, ZFNs are easily packed into a viral vector with limited cargo space, including adeno-associated viral vectors. Furthermore, because ZFN patents expired in 2020, high patent royalties are not required for application. Although functional 6-finger ZFNs can be easily prepared by modular assembly, it has been extremely difficult to produce functional 7-finger ZFNs, which are expected to have higher target specificity than 6-finger ZFNs in some cases. Herein we describe the construction of 7-finger ZFNs and the improvement in genome editing efficiency, which is generally lower in 7-finger ZFNs than in 6-finger ZFNs. Modular assembly of 7-finger ZFNs was achieved using a specific mutation, and the original genome editing efficiency was increased by up to 19%. Furthermore, 7-finger ZFNs showed reduced off-target effects, exhibiting higher target specificity than the corresponding 6-finger ZFNs. Our study provides critical insights for safer and more specific genome editing.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding family of natural products in which biosynthetic maturase enzymes tailor ribosomal precursors into bioactive products. Classical RiPP maturation relies on an N-terminal leader sequence in the precursor peptide and a complementary RiPP-recognition element in the enzyme to guide substrate binding. Herein, we interrogated PapB, a radical S-adenosyl-l-methionine RiPP maturase known to introduce thio-(seleno)-ether cross-links and discovered that its catalytic reach extends well beyond this paradigm. PapB efficiently processes substrates that lack any canonical leader sequence, demonstrating bona fide leader-independent activity. To highlight the practical value of this capability, we applied PapB to three therapeutically relevant analogues of glucagon-like peptide pathway agonists to generate C-terminal cyclic structures. In every case, the enzyme achieved complete conversion of the linear to the thioether macrocyclized peptide. These results establish PapB as a versatile, plug-and-play biocatalyst for late-stage macrocyclization of structurally diverse peptides, opening a direct route to conformationally constrained therapeutic candidates without the need for leader tags.

MraY is an essential bacterial enzyme for peptidoglycan synthesis in cell walls and serves as a promising but unrealized target for developing effective antibacterial drugs. Nature has provided a remarkable array of nucleoside inhibitors of MraY, and researchers have skillfully refined these structures to develop inhibitors that effectively mimic natural products. Yet, both natural products and their synthetic variants often face challenges regarding inadequate in vivo efficacy, and the intricate nature of these structures complicates their synthesis and exploration of structure-activity relationships (SAR). Here, we present our findings on the discovery of first-in-class small molecule MraY inhibitors that are non-nucleoside-derived, based on 1,2,4-triazoles, using a structure-based drug design strategy. By leveraging the structural roadmap of the MraY binding site, we discovered the initial hit compound 1 with an IC₅₀ value of 171 μM in vitro against MraY from Staphylococcus aureus (MraY _SA ) that was refined to compound 12a, exhibiting an IC₅₀ value of 25 μM. Molecular docking studies against MraY _SA provided critical insights into how the binding interactions of compounds directly influence their activity. Furthermore, we report that these compounds show broad-spectrum antibacterial activity against critical pathogens such as Enterococcus spp., methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococci (VRE) strains, Acinetobacter baumannii, and Mycobacterium tuberculosis. This study showcases novel non-nucleoside inhibitors as a compelling proof-of-concept for crafting the next generation of antibacterial agents targeting MraY.

Signal transduction by gaseous small molecules is an essential process in modern living systems, where the gasotransmitters relay cellular signals to bio-(macro)-molecules through covalent bond formation. However, the origin or a primordial version of such signaling events in abiotic worlds has been poorly understood to date. Through examination of chemical reactivities between formaldehyde and cyclic dipeptide/diketopiperazine derivatives in prebiotically relevant solid-state environments, this study demonstrates that the gaseous small molecule may serve not only as a mere building block for the abiotic construction of biomolecules but also as an activating agent that transforms the inert peptide into reactive, prebiotically important chemical species. In addition, superiority of solid-state reactions or mechanochemistry to solution-based reaction conditions described in this article may be an indication of potential significance of the mechanical force-induced chemistry for chemical evolution, in particular for abiotic emergence of polypeptides.

Over the years, the cannabinoid type 1 receptor (CB₁R) has emerged as a promising therapeutic target for addressing various neurodegenerative diseases including HIV-associated neurocognitive disorders (HAND). However, the therapeutic application of direct CB₁R activation is often hindered by undesirable psychoactive side effects. To mitigate this issue, research has focused on utilizing positive allosteric modulators (PAMs) to enhance the CB₁R activity indirectly. Preclinical studies have highlighted the efficacy of CB₁R PAMs, such as ZCZ011 and GAT211, in mouse models of Huntington's disease, neuropathic pain, and, more recently, HAND. Building on this evidence, we employed primary frontal cortex neuronal cultures and whole brain microglial cultures to investigate the direct and indirect effects of racemic ZCZ011 against the HIV-1 trans-activator of transcription (Tat)-induced excitotoxicity. In parallel, molecular modeling and molecular dynamics simulations were conducted using the ZCZ011 enantiomers (R)-ZCZ011 and (S)-ZCZ011, to elucidate their binding profiles at CB₁R. Our in vitro studies revealed that ZCZ011 demonstrated neuroprotective effects against Tat-induced excitotoxicity in the presence of N-arachidonoylethanolamine (AEA) in a dose-dependent manner. Interestingly, racemic ZCZ011 exhibited neuroprotective properties even in the absence of AEA, deviating from the classical behavior of a true PAM. This prompted further investigation into the binding profiles of the enantiomers. Molecular modeling revealed that (R)-ZCZ011 and (S)-ZCZ011 bind to distinct sites on CB₁R, aligning with the binding profiles of other CB₁R allosteric modulators, GAT228 and GAT229. Notably, (R)-ZCZ011 exhibited a higher number of hydrogen bonds and both polar and nonpolar interactions with CB₁R, enhancing the stabilization of AEA binding to CB₁R. In summary, these findings suggest that (R)-ZCZ011 may function as both a PAM and an allosteric agonist, like GAT228, while (S)-ZCZ011 may act as a pure PAM, resembling GAT229. This dual functionality underscores the therapeutic potential of ZCZ011 in modulating CB₁R activity for a number of neuroprotective applications.

The escalating prevalence of antibiotic-resistant bacteria and the increasing complexity of managing severe infections emphasize the critical need for novel and effective antibiotics. Herein, we present a novel computational strategy focused on metal-based antibiotics, specifically rhenium (Re) complexes, for the rational design of next-generation antibacterial agents. Our approach integrates machine learning (ML) classification models to predict antibacterial potency, particularly against multidrug-resistant pathogens. A recognized limitation of conventional ML-driven antibiotic discovery is its dependence on structural similarity to known antibiotics, which hinders the exploration of structurally diverse and innovative antibiotic classes. To address this, we developed predictive ML models based on multi-layer perceptron (MLP) and random forest (RF) algorithms to estimate the minimum inhibitory concentration (MIC) of Re complexes against methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) Staphylococcus aureus strains. Utilizing structural descriptors, these models demonstrated strong predictive performance and were successfully applied to evaluate 26 novel Re complexes. Additionally, Shapley additive explanation (SHAP) analysis provided insights into the structural features influencing antibacterial activity predictions. The study's outcomes affirm the effectiveness of our ML-guided approach as a promising pathway for the rational, de novo design of potent Re based antibiotics capable of combating antibiotic-resistant bacterial infections.

[This corrects the article DOI: 10.1021/acsbiomedchemau.5c00080.].

The sialic acid synthase NeuB, and other α-carboxyketose synthases, are long-standing targets for inhibition by potential antimicrobial compounds. NeuB catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc) that passes through a tetrahedral intermediate (THI) to form N-acetylneuraminic acid. We measured multiple kinetic isotope effects (KIEs) in order to determine the transition state (TS) structure of the NeuB-catalyzed reaction. As part of this study, the use of incomplete T ₁ relaxation during KIE measurement by NMR was investigated, and then used to accelerate KIE measurements 9-fold. KIEs measured at the 3-¹³C, 2-¹³C and 2-¹⁸O positions in PEP reveal a stepwise mechanism of THI formation in which the PEP C3 to ManNAc C1'(aldehyde) bond (C3···C1') is formed first, leading to a cationic intermediate. However, the transition state is so early in C3···C1' bond formation, with a C3···C1' bond order of 0.17 to 0.35, that there is little cationic character in the transition state. The lack of change in geometry or charge in forming the transition state implies that NeuB may function primarily by "catalysis-by-approximation"; i.e., its main contribution to TS stabilization is bringing the reactants together in the correct orientation. It also implies that inhibitors designed to mimic a positive charge are unlikely to be effective. Instead, neutral inhibitors that maximize the number of active site interactions are more likely to be effective.