In 2020 seven genetic variants of the mitochondrial serine hydroxymethyl transferase (SHMT2) were linked to a novel brain and heart developmental syndrome. SHMT2 is a pyridoxal 5′-phosphate (PLP) binding enzyme involved in one-carbon metabolism and mitochondrial redox homeostasis, which also shows several moonlighting functions and protein-protein interactions that are related to its ability to change its oligomeric state. Molecular dynamics suggested that these genetic variants may cause a range of different structural defects ultimately affecting the conformation of the active site, cofactor affinity and the oligomerization state. Here we present a systematic biochemical and structural analysis of these variants. We expressed and purified the SHMT2 mutants and evaluated their oligomeric state, PLP binding affinity, thermal stability, and catalytic activities compared to the wild-type enzyme. The crystal structures of the less active mutants were then solved to pinpoint the structural determinants of the defects highlighted in solution. The results are discussed considering the multi-functional role of SHMT2 and expand our current understanding of the enzyme’s sophisticated conformational and allosteric dynamics.
Antimicrobial resistance (AMR) continues to compromise the clinical utility of existing antibiotics, underscoring the urgent need for alternative discovery strategies that can access novel chemical space. Traditional culture-dependent approaches have reached a plateau, shifting attention toward omics based methods that enable systematic exploration of biologically and chemically diverse natural systems. Extreme ecological niches and medicinal plant-associated environments represent underexplored reservoirs of secondary metabolites shaped by unique selective pressures and chemical interactions, offering promising opportunities for antimicrobial discovery. Metagenomics provides direct access to the biosynthetic potential of uncultured microorganisms, allowing the identification of cryptic biosynthetic gene clusters and previously inaccessible antimicrobial pathways from extreme environments. In parallel, metabolomics facilitates comprehensive profiling, annotation, and functional evaluation of bioactive metabolites derived from medicinal plants and their associated microbiomes. When applied independently, these approaches have expanded the repertoire of antimicrobial candidates; however, their true potential emerges through integrative strategies that connect genomic information with metabolite production and biological activity. This review focuses on recent advances in metagenomics and metabolomics-driven antimicrobial discovery, with particular emphasis on integrative, resistance-aware approaches that bridge genomes, metabolites, and antimicrobial phenotypes. We highlight representative case studies, methodological innovations, and chemical biology perspectives that contribute to the identification of novel antimicrobial agents, targets, and mechanisms of action. Finally, we discuss key analytical challenges and emerging developments, including multi-omics integration, computational tools, and data-driven frameworks, that are shaping future pipelines for antimicrobial discovery. Together, these insights underscore the value of integrated omics approaches in addressing the escalating challenge of antimicrobial resistance.
The GluN1/GluN2A N-methyl-D-aspartate receptor (NMDAR) is a critical ligand-gated ion channel in the central nervous system, playing essential roles in synaptic plasticity, learning, and memory. Understanding its dynamics in the open-channel active state is paramount for deciphering its physiological functions and for developing targeted therapeutics. Despite many past efforts, the active state has not yet been fully resolved at atomic resolutions. To elucidate the molecular mechanism of the NMDAR activation, computer modeling and simulation are instrumental in providing detailed information about the dynamics and energetics of the receptor in various functional states. In this study, we started from a previously built active-like model of a truncated NMDAR (with its ligand-binding domains restrained), and explored its energetics and dynamics with extensive molecular dynamics (MD) simulation (total simulation time is 5.6 µs). Based on the MD simulation, we employed an array of analysis tools to study the fluctuations/motions at the levels of individual residues, the channel pore, and the global structure, and identified a dynamic network of polar/nonpolar interactions between residues. Furthermore, we used machine learning to identify key interactions and residues associated with channel pore opening/closing and annotated them with evolutionary conservation grades and known disease mutations in NMDAR. Taken together, our study provided rich structural/dynamic information and specific predictions for key residues/interactions which will inform future functional studies of the NMDA receptor.
IntroductionMutation-induced drug resistance is a major contributor to the failure of targeted cancer therapies, particularly in tumors driven by mutations in the KRAS oncogene. Although covalent inhibitors effectively target KRAS G12C, secondary mutations such as G12C/Y96C, G12C/Y96S, and G12C/Y96D confer resistance despite leaving the covalent attachment site intact.MethodsTo investigate the conformational basis of this resistance, we developed a computational framework integrating molecular dynamics (MD)-derived structural, energetic, thermodynamic, and contact-based descriptors with machine learning. All simulations were performed in the apo (unbound) state; the results therefore reflect conformational and solvent-exposure correlates associated with resistant mutants rather than inhibitor-specific resistance mechanisms. Molecular descriptors extracted from MD simulations of treatment-sensitive and treatment-resistant KRAS systems were used to train logistic regression, random forest, support vector machine, and Bayesian network classifiers. To address the correlated nature of MD-derived conformers, model performance was evaluated using a system-independent mixed held-out validation scheme, and univariate analysis employed clustering-aware statistical approaches.ResultsCross-referencing machine learning feature importance rankings with within-resistant-group variability testing revealed an important distinction between features reflecting mutant-identity-specific variation and those consistent with a shared resistance phenotype. Residue-level descriptors: solvent-accessible surface area variability at E62 and H95, Lennard-Jones 1,4 interaction energy, and root mean square fluctuation at M72 and H95 were both consistently discriminative across validation schemes and statistically consistent across all three resistant mutants, suggesting that they represent potential apo-state conformational and solvent-exposure signatures associated with resistant KRAS mutants.DiscussionOur proof-of-concept workflow may inform the design of inhibitors targeting secondary KRAS resistance mutations, pending validation in additional structurally independent mutant systems.
Kinetoplastid diseases remain a major global health challenge, highlighting the need for new antiparasitic chemotypes. N,N′-disubstituted aliphatic diamines have emerged as a promising scaffold against trypanosomatid parasites. Building on our previous studies, we expanded the structure–activity relationship (SAR) of this chemotype through the synthesis of fifty-two analogues obtained by one-pot reductive amination of aliphatic diamine linkers with structurally diverse aromatic and heteroaromatic aldehydes. The compounds were first evaluated against the extracellular stages of Trypanosoma cruzi, Trypanosoma brucei, and Leishmania donovani. Several derivatives displayed pIC50 values above 6.0 and Vero-cell selectivity index above 10. Based on these results, nine hits were further evaluated against the promastigote and amastigote stages of L. amazonensis, L. braziliensis, and L. infantum, responsible for cutaneous and visceral leishmaniasis. Among them, compounds 12a, 12m, and 8e showed potent activity against intracellular amastigotes with selectivity indices above 10, highlighting this scaffold as a promising starting point for antileishmanial drug discovery.
IntroductionAmyotrophic lateral sclerosis (ALS) is a rapidly progressive and always fatal disease that is associated with the selective death of upper and lower motor neurons, and for which the molecular basis of disease causation by mutations identified in affected individuals is still poorly understood. A large proportion of ALS is associated with mutations of genes that code for RNA-binding proteins, and notably mutations in the gene that codes for FUS (Fused in Sarcoma), a highly multifunctional protein that participates in RNA biology and metabolic processes that is highly characterized by its intrinsically disordered regions.MethodsAs part of this dissertation research, the structural and functional implications of three different mutations associated with ALS in the FUS protein (G156E, R234L, and R521C) that map to distinct functional regions of the protein were analyzed by a computational scheme that integrates structure prediction by AlphaFold, calculation of intrinsic plasticity and binding plasticity by FuzPred, and molecular modeling simulations by HADDOCK to study crucial protein-protein and protein-RNA interactions.ResultsThese studies clearly show that, while there is no large structural change in the overall protein structure, each of the mutations causes a site-directed and functionally relevant alteration in conformational dynamics and protein-binding properties. In particular, the R521C mutation enhances the difference in binding affinity between the FUS Nuclear Localization Signal (NLS) and Transportin-1, indicating that there may be a mechanistic cause for impaired transport, whereas the G156E mutation destabilizes FUS-RNA binding, indicating that there may be a change in RNA-binding affinity.DiscussionThe results confirm that ALS-causing mutations in FUS cause disease primarily as a result of subtle changes in intrinsic disorder, binding versatility, or molecular recognition, as opposed to classical protein misfolding and that structural biology predictions have provided insights that have helped to further our understanding of ALS.
Mycolic acids (MAs), which are β-hydroxy fatty acids with long alpha-alkyl side chains, are major components of the cell wall of Mycobacterium tuberculosis (Mtb), which causes tuberculosis. These lipids also occur in M. tuberculosis var bovis BCG (Bacillus Calmette-Guérin), a vaccine strain. Ongoing research, that followed observations on the off-target beneficial effects of BCG vaccination has raised a possibility that methoxy- and keto-derivatives of MAs (M-MAs and K-MAs) may help to train human innate immune cells to combat and prevent sepsis, a life-threatening condition resulting from uncontrolled spread of microbial and viral infections. To allow facile testing of this possibility, we have developed methods for the generation of M-MAs and K-MAs without using Mtb, a pathogen, or BCG, which grows slowly. We have developed methods for facile production of substantial amounts of M-MAs and K-MAs by engineering Mycobacterium smegmatis (Msm), a non-pathogenic and fast-growing organism, to express MA-oxygenation genes from Mtb, and purifying these MAs from the recombinant strains (approximately 70 mg from a 100 mL culture); the authenticities of the products were confirmed via mass spectrometry. The system could also provide additional oxygenated MAs, epoxy- and hydroxy-derivatives. This advancement will help to interrogate the possible application of oxygenated mycolic acids in treating sepsis in humans with a manageable cost and relative ease. This applied study has also provided leads for foundational studies on how individual oxygenation and cyclopropanation system influences each other in terms of product formation.
Zinc is an essential trace element that plays a critical role in synaptic transmission, neuronal survival, gene regulation, and antioxidant defence in the brain. Hence, one of the central contributors to Alzheimer’s disease (AD) pathology is the disruption of zinc homeostasis. Recent studies suggest that an interconnected regulatory network maintains neuronal stability, comprising zinc-binding proteins, such as metallothioneins, zinc-dependent enzymes, zinc finger transcription factors, and zinc transporters. The accumulation of amyloid-β, Tau hyperphosphorylation, mitochondrial dysfunction, oxidative stress, and chronic neuroinflammation are the underlying causes of the mislocalization of zinc in AD. Also, the involvement of zinc-associated enzymes influences the amyloid clearance, as well as transcriptional regulation, which disrupts neuronal proteostasis. Hence, the therapeutic strategies usually aim at restoring the zinc balance, which involves the small-molecule metal modulators, natural phytochemicals with zinc-interacting properties and gene-based approaches. Hence, understanding zinc-binding protein networks provides a system-level framework for developing multi-target therapeutic interventions that could slow rapid progression, as well as might increase neuronal resilience in AD.
Over the last 30 years, many studies have examined polyoxidometalates as a treatment for various illnesses. Many substances that contain decavanadate have been proposed as potential therapies for diabetes, cancer, and Alzheimer's disease. Currently, there are 8 functional proteins in the Protein Data Bank (PDB) that bind decavanadate (V10). These are the human cell cycle protein CksHs1, the transient receptor potential cation channel (TRPM4), two ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), acid phosphatase, tyrosine kinase, and, more recently, HEWL (lysozyme) and RNase A. The interaction sites of the decavanadate anion with these proteins are mostly made up of side chains, such as arginine, lysine, and histidine. The histaminium dication served as a small biomimetic model to elucidate the noncovalent interactions between decavanadate and histidine residues in proteins. Here we report the synthesis and crystallization of histaminium decavanadate and its characterization using FTIR, Raman, 51V-NMR, TGA, and X-ray diffraction. To gain insights into the non-covalent interactions of decavanadate in protein environments, the interaction energy between the decavanadate anion and histaminium counterions was calculated using DFT methods. To better understand the non-covalent interactions in these adducts, studies were carried out using the Quantum Theory of Atoms in Molecules (QTAIM) and Non-covalent Interaction-Reduced Density Gradient (NCI-RDG). Based on their interaction energies, decavanadate can interact with histaminium through hydrogen bonds, electrostatic forces, and van der Waals forces. The compound exhibits strong hydrogen bond interactions, which significantly enhance its stability. Decavanadate holds considerable promise as a potential treatment for diabetes, cancer, and neurological protection. Consequently, additional research is required to elucidate its interactions with proteins.
Cannabinoid receptors 1 (CB1) and 2 (CB2) are key components of the endocannabinoid system and play central roles in regulating diverse physiological processes, including neural signaling, immune modulation, and inflammation. Both receptors belong to the Class A G protein-coupled receptor (GPCR) family and exhibit distinct tissue distributions and signaling profiles, making them attractive yet challenging targets for receptor-specific drug development. Efforts to design subtype-selective ligands have been hindered by the high degree of sequence and structural similarity between CB1 and CB2, resulting in widespread ligand cross-reactivity. Recent advances in structural biology have yielded high-resolution structures of both CB1 and CB2 in multiple functional states, providing new opportunities to dissect the molecular determinants of receptor selectivity. In this review, we present a comprehensive comparative analysis of CB1 and CB2 structures, with a focus on differences in ligand-binding residues, binding pocket topology, and physicochemical environments that may influence subtype selectivity and signaling. Our analysis suggests that CB1 preferentially accommodates bulkier and more flexible ligands, whereas CB2 favors smaller and more compact chemotypes. In addition, positionally conserved but chemically distinct residues (e.g., Leu193 in CB1 vs. Ile110 in CB2 and Leu359 in CB1 vs. Val261 in CB2) highlight subtle structural differences that can influence ligand binding and receptor activation. Overall, this work aims to inform the rational design of next-generation cannabinoid ligands with enhanced receptor selectivity, reduced side effects, and improved therapeutic potential.
Xylazine, a non-opioid α2-adrenoceptor agonist, is increasingly implicated in misuse and opioid-adulterated overdoses. Tolazoline, a non-selective α-adrenergic antagonist, is widely used in veterinary medicine to reverse xylazine-induced sedation and cardiovascular depression. Here, we combined molecular docking, molecular dynamics simulations, and in silico ADME (absorption, distribution, metabolism, and excretion)/Tox predictions to elucidate the pharmacological interplay between xylazine and tolazoline. Both compounds displayed comparable binding energies and stable interactions at the serotonin 5-HT7 and κ-opioid receptors, supporting a competitive mechanism at shared receptor sites. Comparative in silico ADME profiling revealed that xylazine exhibits high blood–brain barrier penetration, extensive plasma protein binding, and rapid clearance, favouring potent but short-lived central nervous system effects. Conversely, tolazoline was predicted to demonstrate high lipo-solubility levels, low protein binding, large unbound fraction, and long half-life, enabling sustained peripheral α-blockade and sufficient central penetration to counteract xylazine’s sedative and sympatholytic actions. These complementary pharmacokinetic and pharmacodynamic features suggest a mechanistic rationale for tolazoline’s clinical efficacy as an antidote. By integrating receptor-level interactions with kinetic and distributional properties, our findings offer novel insights into the reversal of xylazine intoxication and generate testable predictions for transporter-mediated dynamics and PK/PD (Pharmacokinetic/Pharmacodynamic) modeling.
Stimulated Raman scattering (SRS) microscopy is a fast Raman imaging technique that combines the molecular specificity of vibrational spectroscopy with the high spatial resolution and speed of laser-scanning microscopy. Building on the historical development of Raman and coherent Raman scattering (CRS) theories, advances in ultrafast lasers, modulation schemes, and detection electronics over the past 2 decades have transformed SRS from a laboratory curiosity into a practical platform for chemical imaging. This review article provides an integrated overview of the fundamentals of Raman spectroscopy and SRS microscopy, and presents the architecture of modern SRS instruments, including single-band and hyperspectral designs, contrast mechanisms, and the use of Raman probes. It also reviews recent technological progress in fiber laser sources, denoising and high-sensitivity detection schemes, hybrid and endoscopic SRS implementations, and emerging quantum-enhanced SRS (QE-SRS) approaches that aim to push sensitivity beyond classical limits. On the applications side, this review highlights label-free metabolic imaging at the single-cell level, bioorthogonal Raman tagging of drugs and metabolites, and tissue studies that link lipid metabolism to disease. A dedicated section summarizes the development of stimulated Raman histology (SRH) for intraoperative rapid diagnosis and surgical guidance, including deep-learning convolutional neural network (CNN) and artificial intelligence (AI) models that enable near real-time interpretation of fresh brain and other neoplastic tissues. Additional topics covered in this review include drug delivery, environmental and materials science, analysis of micro- and nanoplastic particles (MNPs), and imaging of fungal, bacterial, and plant systems. Taken together, the studies summarized in this review show that SRS microscopy has matured into a versatile and reliable modality for non-fluorescent chemical imaging across biology, medicine, and materials science, while continued progress in laser sources, detection, computation, and probe design is expected to further expand its capabilities and impact.
The active components of the turmeric rhizome, primarily curcumin, have demonstrated remarkable pharmacological activity against a wide range of diseases. Among its most notable properties are its anti-Alzheimer’s, anti-arthritic, anti-cancer, antidepressant, anti-diabetic, anti-inflammatory, antimicrobial, anti-osteoporotic, antioxidant, hepatoprotective, neuroprotective, renoprotective, and immunomodulatory activities. Furthermore, curcumin is a highly bioactive natural compound that exhibits multiple biological interactions and therapeutic targets, possesses fluorescent properties, and shows low toxicity even at high doses. However, its medical application is limited by several physicochemical and pharmacokinetic disadvantages, including low solubility and bioavailability, hydrophobicity, rapid metabolism and systemic elimination, and low absorption and high photosensitivity. A promising strategy to overcome these limitations involves coordinating curcumin with metal ions through its β-dicarbonyl fragment, allowing it to act as a bidentate ligand. The formation of metal complexes can improve the solubility, bioavailability, and cellular uptake of curcumin, enhancing its pharmacological, chemopreventive, and therapeutic activities compared to the free molecule and its uncoordinated derivatives. In particular, coordination with second- and third-row transition metals (4d and 5d series) is especially attractive due to the high stability of their complexes under physiological conditions. Numerous curcumin complexes with these metals have been described; however, there is still a notable lack of representatives of some elements, such as molybdenum and osmium. To date, the most studied biomedical applications for these complexes include antibacterial, anticancer, and antifungal activities, although anti-arthritic, anti-inflammatory, anti-osteoporotic, antioxidant, anti-rheumatic, and antiviral properties have also been investigated. Therefore, curcumin complexes with second- and third-row transition metals represent a promising platform for the development of new therapeutic agents, and increasing interest in their study is expected to define their potential preventive and medical applications in the future.
Photobleaching impedes the application of fluorescent dyes in long-term imaging. While enzymatic oxygen scavenging enhances photostability, it induces progressive acidification of the solution. Here, we report a new oxygen-scavenging system comprising squalene (SQ) and Trolox that improves dye stability without perturbing pH. DPPH and ABTS radical cation scavenging assays verified the antioxidant activity of SQ, with 0.7–1% concentrations achieving 35% radical scavenging capacity. The system maintains pH stability for more than 18 min, in contrast to the rapid acidification observed with glucose oxidase/catalase. Total internal reflection fluorescence (TIRF) imaging of cyanine 3 (Cy3) and cyanine 5 (Cy5) revealed that SQ with Trolox performs comparably to enzymatic systems while permitting extended illumination, establishing a pH-neutral strategy for enhanced photostability.
Herein, we present two novel heterobimetallic complexes Ru(II)-Mn(II) (Ru(η6-p-cymene)Cl2PCpMnCl2(H2O)2 (RuPCpMn) and Ru(η6-p-cymene)Cl2PNrMnCl2(H2O)2 (RuPNrMn)) with phosphines (PCp, PNr) derived from fluoroquinolones antibiotics (HCp–ciprofloxacin and HNr–norfloxacin). Compounds were characterized using elemental analysis, Fourier-transform infrared spectroscopy (FT-IR), electron paramagnetic resonance (EPR), electronic absorption spectroscopy (UV-Vis), and electrospray ionization mass spectrometry (ESI-MS). The antibacterial activity in vitro of heterobimetallic complexes together with literature monometallic CuI and RuII complexes possessing the same and different phosphine ligands was evaluated. The results indicate that structural modifications of metal-fluoroquinolone complexes affect their antibacterial properties. The mononuclear ruthenium(II) complexes exhibited broad-spectrum activity, particularly against Gram-negative bacteria, whereas the incorporation of manganese(II) ions did not lead to substantial improvement of antibacterial activity in vitro. However, complexes containing phosphine ligands without fluoroquinolone motifs but with, e.g., alkaloid, exhibited significantly reduced activity, emphasizing the importance of maintaining the pharmacophoric integrity of the fluoroquinolone core.
The hybridized mixed-valence isopolyoxomolybdate [N(CH3)4]5 [MoV10MoVI3(OH)2O37(C4H4NO2)]·6MeOH (IDA-MoV10MoVI3) was synthesized through a solvothermal one-pot method in a non-aqueous environment. The isopolyoxomolybdate scaffold was hybridized with iminodiacetic acid (IDA) by chelating a terminal molybdate group. The structural integrity and composition of IDA-MoV10MoVI3 were confirmed through comprehensive analyses, including single-crystal X-ray diffraction (XRD), elemental analyses, infrared (IR) spectroscopy, Resonance Raman (RR) spectroscopy, UV-vis spectroscopy, and thermogravimetric analysis (TGA). Bond-valence-sum calculations (BVS) provided evidence that (IDA-MoV10MoVI3) is approximately 77% reduced. The high degree of reduction unambiguously places IDA-MoV10MoVI3 in the ‘molybdenum-red’ family of highly reduced isopolymolybdates. Initial antiviral and cytotoxicity assays demonstrated good biocompatibility of IDA-MoV10MoVI3, with no observable toxicity and no significant antiviral activity up to 200 µM.
CDK5 is an atypical member of the CDKs family that is not directly involved in cell cycle regulation but has a very relevant role in promoting DNA damage response and immune response evasion in cancer. Its action in these tasks conflicts with those of other CDKs, namely CDK4 and CDK6. It is therefore relevant to find small molecule catalytic inhibitors that could be selective for CDK5. Roscovitine has been found to inhibit CDK5 more efficiently than CDK4 and CDK6 in vitro, appearing to be an ideal initial candidate. We analyzed the structural interaction of Roscovitine and some of its derivatives with different CDKs by molecular docking, dynamics and free energy calculation to identify those showing a high affinity for CDK5 (and eventually CDK2) accompanied by optimal discrimination against CDK4/CDK6 and to derive chemical clues to eventually produce other, more selective and efficient compounds.
Zinc-dependent aminopeptidases are a subclass of exopeptidases implicated in the hydrolysis of N-terminal residues from peptides through a Zn(II) co-factor dependent hydrolytic mechanism. In humans, the prominent members of this class of enzymes (ERAP1/2, IRAP, APN/CD13, APA) catalyse peptide trimming in antigen presentation, peptide hormone regulation, and peptide homeostasis. Since the catalytic activity of these enzymes relies on a tightly coordinated Zn(II) ion in their conserved H-E-X-X-H…E motif, they constitute attractive yet challenging therapeutic targets. In this review, I provide an updated survey of their structural and mechanistic principles, evaluate their physiological and pathological roles, and outline emerging strategies for selective modulation and biotechnological usage. I also discuss current obstacles and future directions in deploying zinc-targeted aminopeptidase chemistry in translational settings.
