Search Results (422)

1-Deoxynojirimycin (DNJ), a well-characterized α-glucosidase inhibitor, remains an important target for dose-reduction and formulation strategies. In the present study, we evaluated the individual and combined α-glucosidase inhibitory activities of nine dietary flavonoids with DNJ, quantified synergistic effects using the combination index (CI) method, and elucidated the molecular mechanism through integrated enzyme kinetics, multi-spectroscopic techniques and molecular docking. (+)-Catechin exhibited the strongest inhibitory activity (IC₅₀ = 33.7 ± 2.7 μM), and its combination with DNJ produced synergistic inhibition across all doses (CI < 0.7). Kinetic analysis confirmed that DNJ acted as a competitive inhibitor, while (+)-catechin functioned as a non-competitive inhibitor. Fluorescence quenching assays revealed that (+)-catechin pre-incubation increased the binding affinity of DNJ to α-glucosidase by 393%. Circular dichroism spectroscopy showed that (+)-catechin induced a marked β-sheet-to-α-helix conformational conversion, and co-incubation of both inhibitors produced more secondary structural changes than either inhibitor alone. Molecular docking further confirmed their distinct binding sites. These findings demonstrate that (+)-catechin synergistically potentiates DNJ activity through allosteric conformational modulation, providing an experimental basis for optimizing DNJ-containing formulations. Full article

Glioblastoma multiforme is the most aggressive primary brain tumor in adults, which displays extremely poor prognosis. Protease-activated receptor 2 (PAR2) and its downstream effector SerpinB3 are overexpressed in aggressive glioblastomas. In this study we evaluated the antitumor activity of 1-piperidine propionic acid (1-PPA), an allosteric PAR2 inhibitor, in in vitro preclinical models of glioblastoma. PAR2 and SerpinB3 were analyzed at the transcriptional and protein level in glioblastoma cell lines and primary cultures. These were treated with 1-PPA alone or in association with temozolomide (TMZ) and the effects evaluated by Incucyte^® technology. Pharmacokinetics and tissue distribution of 1-PPA were assessed in mice by LC-MS/MS. 1-PPA significantly reduced glioma cell proliferation, migration, and invasion, thus promoting apoptotic cell death, in a concentration-dependent manner. The combined treatment with TMZ led to a concentration-dependent decrease in cell proliferation (12–20%) compared to TMZ alone. Molecularly, 1-PPA downregulated PAR2 and SerpinB3 expression. Pharmacokinetic studies in healthy mice showed that 1-PPA is systemically bioavailable and distributes to several organs, including the brain. These data indicate that 1-PPA shows brain exposure and capability to affect different hallmarks of aggressiveness in glioblastoma cells, including hyperproliferation and invasion, supporting its further development as a novel therapeutic strategy in these tumors. Full article

Staphylococcus aureus (S. aureus) remains a major global pathogen and a significant public health concern due to its antibiotic resistance. This has spurred the search for new treatments, resulting in the discovery of two promising targets: FtsZ and FabI. Naturally occurring compounds berberine and lipophilic acids are known to bind these enzymes, respectively. This study aims to improve berberine’s binding affinity for FtsZ and enhance its interaction with FabI by designing hybrid compounds that could serve as dual inhibitors, targeting both active and allosteric sites. Forty-eight hybrids, derived from berberine and lipophilic acids with 10 to 22 carbons, were modeled. Molecular docking against five S. aureus enzyme crystal structures identified six compounds with geranic acid chains (1s, 1t, 1u, 2s, 2t, 2u) that showed the strongest binding. Among these, 2s, 2t, and 1t showed the greatest affinity for FtsZ, while 2u, 1u, and 1s targeted FabI, with binding energies around −8.2 to −10.5 kcal/mol. QSAR models estimated MICs within known inhibitor ranges, implying potential effectiveness. Hydrophobic and flexible features correlated with stronger interactions and activity. ADMET analysis indicated low toxicity for these hybrids. Modifying berberine with lipophilic acids appears to be a promising approach for developing plant-based dual inhibitors against S. aureus. Full article

Background: Combining natural products with conventional antidiabetic agents to inhibit α-glucosidase activity is an effective strategy for preventing postprandial hyperglycemia. Baicalein, a natural flavonoid with well-documented low toxicity, showed potential synergistic effect with acarbose in diabetic models; however, the synergistic performance and mechanisms of the two agents targeting human maltase–glucoamylase (MGAM) remain unclear. Methods: Recombinant human MGAM-C and MGAM-N were expressed in Pichia pastoris for in vitro inhibition assays. Maltose-loaded mice were used to assess the in vivo hypoglycemic activity and intestinal maltase inhibition. Inhibitor–enzyme interactions were investigated by fluorescence spectroscopy, circular dichroism (CD), multiple molecular docking, and molecular dynamics (MD) simulations. Results: Baicalein potently inhibited MGAM-C and MGAM-N with IC₅₀ values of 20.41 ± 4.80 μM and 14.04 ± 0.94 μM, respectively, and demonstrated a synergistic effect when combined with acarbose. In vivo, co-administration significantly reduced blood glucose levels and suppressed small intestinal maltase activity in maltose-loaded mice. Mechanistic studies revealed that baicalein functions as a non-competitive inhibitor by binding to the allosteric site of MGAM-C via stable hydrogen bonds with residues Ile1716 and Trp1749. This interaction induces conformational changes in the enzyme’s secondary structure and optimizes the hydrophobic microenvironment of the active site, thereby enhancing the binding affinity and hydrogen bond stability of acarbose. These molecular events collectively contribute to the synergistic inhibition of MGAM-C hydrolytic activity. Conclusions: This research revealed the synergistic inhibitory effect of baicalein and acarbose on MGAM and the underlying mechanisms, thereby providing a theoretical basis for developing pharmaceutical formulations to enhance acarbose efficacy. Full article

Background: Cyclin-dependent kinase 4 (CDK4) is a key regulator of cell-cycle progression and an established therapeutic target for breast cancer. Although the unique architecture of its ATP-binding site has enabled the development of highly selective inhibitors, the emergence of acquired resistance highlights the need for alternative therapeutic strategies targeting protein conformational regulation. Methods: The conformational landscape of CDK4 was investigated in this study using accelerated molecular dynamics (aMD) simulations combined with Markov state model (MSM) analysis to identify cryptic conformational states and potential allosteric binding sites. Particular attention was given to the glycine-rich loop (G-loop), a critical structural element that shapes the ATP-binding pocket, and to the effects of compound 8i (1-(2-(3-chlorobenzoyl)hydrazine-1- carbonyl)-N-(pyridin-3-yl)pyrrolidine-2-carboxamide), previously synthesized in our laboratory, on CDK4 dynamics. Results: A distinct conformational transition was identified in which the G-loop shifted toward the N-terminus, resulting in the exposure of a previously unrecognized allosteric pocket adjacent to the catalytic site. Compound 8i interacted with Leu147 and was associated with stabilization of conformational states that favor exposure of the cryptic pocket. Conclusions: These observations suggest that ligand binding may modulate the conformational landscape of CDK4 and favor formation of a cryptic pocket with potential allosteric characteristics. The identified conformational mechanism provides new insights into the dynamic regulation of CDK4 and suggests that stabilization of transient allosteric states represents a promising strategy for the rational design of next-generation CDK4 inhibitors with the potential to overcome resistance in breast cancer therapy. Full article

Octyl (N-(5-(1H-indol-2-yl)-2-methoxyphenyl)sulfamoyl)carbamate has previously been characterized as substrate-specific inhibitor of the linoleic acid oxygenase activity of mammalian ALOX15 orthologs. Here we aimed at optimizing the inhibitory properties of this compound by three different chemical modifications: (i) replacement of the indole core by a phenylpyrrole; (ii) introduction of hydrophilic residues into the aliphatic hydrocarbon chain of the lead compound or by replacing this building block by a triethylene glycol moiety; (iii) replacement of the sulfamoylcarbamate group by a sulfonamide. The inhibitory potencies of the modified compounds for pure rabbit ALOX15 were quantified by in vitro inhibitory assays, and our data indicate that the replacement of the rigid indole core induced a partial loss in the inhibitor’s potency. The introduction of a hydrophilic group into the aliphatic hydrocarbon chain or its replacement by a triethylene glycol moiety improved the solubility of the compound in aqueous solution, but reduced the inhibitor potency by more than one order of magnitude. Finally, the replacement of the sulfamoylcarbamate moiety by sulfonamide improved the substrate selectivity of the inhibitor for rabbit and human ALOX15. The new compounds were highly potent for human and rabbit ALOX15, but did not inhibit human ALOX15B and were less effective for mouse Alox15 (ortholog specificity). Full article

Diabetes mellitus, particularly type 2 diabetes mellitus (T2DM), represents a major global health challenge, driven by the increasing prevalence of obesity and sedentary lifestyles. T2DM is characterized by insulin resistance and progressive β-cell dysfunction, leading to chronic hyperglycemia and multiple complications. Among the molecular targets investigated for therapeutic intervention, protein tyrosine phosphatase 1B (PTP1B) has emerged as a key negative regulator of insulin signaling. By dephosphorylating the insulin receptor and its downstream substrates, PTP1B attenuates insulin action and contributes to metabolic dysfunction. In addition to its role in glucose homeostasis, PTP1B is implicated in obesity, diabetic complications, neurodegenerative disorders, and cancer, highlighting its relevance as a multifunctional therapeutic target. However, the development of PTP1B inhibitors remains challenging due to the highly conserved and polar nature of its catalytic site, which limits selectivity and cell permeability. Recent research has focused on alternative strategies, including allosteric modulation and multi-site inhibition, to overcome these limitations. This review provides a comprehensive overview of PTP1B inhibitors from both synthetic (2019–2025) and natural sources, with particular emphasis on natural products reported from 2022 onwards, while including selected earlier studies to provide historical context and illustrate representative structural classes and inhibition mechanisms. Although PTP1B remains an attractive therapeutic target, its clinical validation for diabetes treatment has yet to be achieved. Continued advances in medicinal chemistry and allosteric modulation may help overcome the current translational barriers. Full article

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), members of the family Pneumoviridae, represent a foremost global cause of acute lower respiratory tract infection in infants, young children, the elderly, and immunocompromised individuals. Despite the recent approval of preventive vaccines and monoclonal antibody prophylactics targeting the viral fusion protein, no widely adopted, RSV-specific direct-acting antiviral is currently approved for routine post-infection treatment. The large (L) protein of the viral RNA polymerase complex, which catalyzes genome replication and mRNA transcription in concert with its obligate cofactor, the phosphoprotein (P), constitutes an ideal drug target owing to its essential and multifunctional enzymatic activities and its absence from host cells. Over the past decade, Cryo-electron microscopy (Cryo-EM) has yielded a series of landmark structures of Pneumoviridae L–P complexes, including apo forms of RSV (at 3.2–3.67 Å) and hMPV (at 3.7 Å) polymerases, among the first promoter-bound non-segmented negative-sense (nsNSV) RNA virus polymerase structures (at 3.40–3.41 Å), and inhibitor-bound complexes that illuminate the molecular basis of non-nucleoside inhibitor (NNI) action at sub-nanomolar potency. This review synthesizes the structural biology of Pneumoviridae RNA polymerases from a chronological and mechanistic perspective, compares RSV and hMPV L protein active sites at near-atomic resolution, and critically evaluates how structural insights are being translated into next-generation antiviral drug candidates, including nucleoside analog inhibitors, allosteric non-nucleoside inhibitors, and emerging candidates at various stages of preclinical and clinical investigation. Full article

Background: The dysregulation of the CDK1/Cyclin B1 complex drives tumor progression in breast cancer (BC). The natural flavonoid luteolin (LT) shows anti-cancer potential, but its mechanism targeting CDK1/CCNB1 remains unclear. Methods: CDK1, CCNB1, and CCNB2 expression were profiled in normal and BC cell lines. An engineered HEK293T GST-CDK1/CCNB1 cell model was used to evaluate LT’s effects on proliferation, ROS levels, and target gene transcription. Computational approaches (molecular docking, dynamics simulations, pharmacophore modeling, MM/GBSA, ADMET, and network pharmacology) assessed LT and its analogues. Results: CDK1/CCNB1 expression was lower in MCF7 BC cells than in normal cells, suggesting the loss of a growth barrier. In engineered HEK293T cells, LT suppressed CCNB1 transcription with minimal effect on CDK1 levels, correlating with anti-proliferative and ROS-modulating effects. Computational analyses confirmed stable LT binding to the CDK1/CCNB1 complex. Designed LT analogues showed improved binding and favorable ADMET profiles. Network pharmacology identified cell cycle regulation, particularly in BC stem cells, as the primary pathway targeted. Conclusions: LT and its analogues inhibit the CDK1/Cyclin B1 complex, revealing a dual mechanism that suppresses both tumor growth and BC stemness. Full article

Selective inhibition of MEK isoforms remains a central challenge in MAPK-targeted drug discovery, largely due to the structural similarity between MEK1 and MEK2. While MEK1 has been extensively characterized, the structural basis of MEK2-specific ligand recognition is not fully understood. Here, we present crystal structures of human MEK2 in complex with the noncompetitive inhibitor U0126 and the allosteric inhibitor refametinib at resolutions of 3.15 Å and 3.30 Å, respectively. Despite a conserved kinase fold, MEK2 exhibits isoform-specific features within the N-lobe β-sheet. Additional differences are observed in the relative orientation of the helix C and activation segment, and the helix F-supported regulatory spine. Structural differences are reflected in micromolar binding affinities for U0126 (K_d = 9.8 μM) and refametinib (K_d = 7.4 μM). Notably, a single N-lobe substitution (Thr87 in MEK2 versus Phe83 in MEK1) selectively enhanced U0126 binding. The MEK2 T87F mutant exhibited an approximately twofold increase in affinity, while refametinib binding remained largely unchanged. SEC–MALS analysis demonstrated that MEK2 predominantly exists as a monomer in solution, contrasting with the reported homodimeric behavior of MEK1. Molecular dynamics simulations supported these findings by revealing isoform-specific differences in oligomeric state-dependent flexibility and inhibitor-induced dynamics. Collectively, our findings define the structural basis underlying the differential inhibitor recognition of MEK2 and MEK1, providing mechanistic insight into isoform-selective MEK-targeted drug design. Full article

Background/Objectives: First-generation CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) target the conserved ATP-binding pocket of CDK4 and, despite clinical success, are limited by acquired resistance and insufficient exploration of alternative regulatory sites. This study aimed to identify a putative allosteric small-molecule candidate at the CDK4 αE-helix–Cyclin D1 α1-helix protein–protein interaction (PPI) interface within the CDK4/Cyclin D1/p21 ternary complex using RapidFunnel-AI, a decision-interpretable virtual-screening pipeline. Methods: Starting from 50,000 ChEMBL 33 molecules, the pipeline sequentially applied a Q-Fold/RapidFunnel topological Tanimoto scan based on clinical CDK4/6 inhibitor motifs, fragment-level electronic-property enrichment, ADMET/PAINS filtering, dry Vina-GPU docking, hydration-mediated AutoDock-GPU (Version 1.6) docking, explicit-solvent molecular dynamics, contact-retention analysis, and MM-GBSA energy decomposition. The Q-Fold Thermo-Core surrogate model provided fragment-level enrichment, predicting the HOMO–LUMO gap (R² = 0.93) and isotropic polarizability (R² = 0.98) on QM9. Candidate selection did not rely on the lowest docking or MM-GBSA score alone, but on pose persistence, contact continuity, and energy-component consistency. Results: The workflow reduced the initial library to 43 topologically prioritized candidates, 25 ADMET/PAINS-filtered ligands, and 9 docking-derived complexes for MD validation. Ligand_020 emerged as the only candidate that preserved a persistent binding mode at Site 2 during a 500 ns simulation—an interface engagement reproduced across three independent 500 ns replicates with no full dissociation in any replicate—with a protein Cα RMSD of 2.88 ± 0.32 Å, a ligand heavy-atom RMSD of 3.56 ± 0.28 Å, and a van der Waals-dominated MM-GBSA profile (ΔGbind = −28.23 ± 3.57 kcal/mol). In contrast, palbociclib and ribociclib, forcibly placed at Site 2 as negative controls, lost most initial contacts within 5 ns and tended to detach despite more favorable MM-GBSA values. Conclusions: These results suggest that single-score docking or MM-GBSA ranking can generate false positives at shallow PPI interfaces. By integrating AI-assisted prioritization, multipocket docking, explicit-solvent MD, contact-retention analysis, and energy-component consistency, RapidFunnel-AI nominated Ligand_020 as an experimentally testable putative allosteric hit targeting the CDK4/Cyclin D1 interface, offering a reusable platform for PPI-focused oncological drug discovery. Full article

Ribosome-inactivating proteins are a class of toxins that target eukaryotic ribosomes, inhibit protein synthesis, and ultimately induce cell death. Several of these toxins pose significant clinical and public health threats. Among these, ricin, derived from the castor bean plant (Ricinus communis), is a highly potent biotoxin with recognized bioterrorism potential. Other ribosome-inactivating proteins, including Shiga toxin produced by pathogenic Shigella and Escherichia coli, as well as mucoricin from Mucorales fungi, contribute to disease severity and can lead to life-threatening complications. Despite these risks, no approved therapeutics are currently available. The development of effective inhibitors depends on robust and well-defined strategies to identify and validate small molecules that disrupt toxin–ribosome interactions. Efforts to target the catalytic active site have met with limited success, largely due to its broad, shallow, and highly polar architecture, which is not conducive to high-affinity binding by drug-like molecules. In contrast, the ribosome-binding interface represents a more tractable target, as it is essential for toxin recruitment and offers more structurally defined and druggable features. Inhibitors targeting this interface can also exert allosteric effects by disrupting long-range conformational coupling between the ribosome-binding region and the active site, thereby attenuating catalytic activity without directly engaging the catalytic pocket. In this review, we compile and evaluate biophysical and biochemical assays for the discovery and characterization of small-molecule inhibitors that target toxin–ribosome interactions. We examine in vitro binding approaches, including surface plasmon resonance-based fragment screening and fluorescence anisotropy assays for ranking inhibitory activity. We further review biochemical and molecular assays that assess ribosome protection from toxin-mediated depurination, along with complementary cell-based assays that evaluate functional rescue in cellular systems. Collectively, this review consolidates current screening methodologies and highlights opportunities to refine assay strategies, thereby supporting the advancement of targeted therapeutics. Full article

Janus kinase 2 (JAK2) occupies a central position in cytokine signaling and plays essential roles in hematopoiesis, immune regulation, and cancer. Although recent advances in structural biology, cryo-EM, receptor modeling, and biophysical analysis have substantially expanded current views of JAK2 function, key mechanistic questions remain regarding how receptor geometry, JH2-mediated autoinhibition, and disease-associated mutations are structurally integrated. In this review, we discuss the multidomain organization of JAK2 and examine how the FERM–SH2 module, the pseudokinase domain (JH2), and the catalytic kinase domain (JH1) cooperate to govern receptor specificity, allosteric control, and cytokine-induced activation. We further analyze how pathogenic mutations rewire this regulatory system by weakening autoinhibitory contacts, altering linker-mediated communication, or stabilizing active dimeric conformations. Finally, we assess current and emerging therapeutic strategies, from ATP-competitive inhibitors to macrocyclic and JH2-selective allosteric modulators, with emphasis on how structural insight can guide next-generation drug design. These advances support a more integrated view of JAK2 regulation and define new opportunities for selective therapeutic intervention. Full article

Chronic myeloid leukemia (CML) represents a paradigm of targeted therapy, driven by the BCR::ABL1 fusion kinase. Over the past four decades, therapeutic strategies have evolved from early molecular targeting approaches and interferon-α to tyrosine kinase inhibitors (TKIs), dramatically improving survival and transforming CML into a largely controllable disease. To provide a comprehensive overview of this evolution, we conducted a narrative literature search across the PubMed and Embase databases, selecting peer-reviewed articles, international guidelines, and landmark clinical trials based on their historical and clinical relevance. Through this expert-driven synthesis, focusing on key milestones in CML therapy, including antisense strategies, interferon-based treatment, first-, second-, and third-generation TKIs, and the development of allosteric inhibitors, this paper analyzes current management strategies, treatment-free remission (TFR), and emerging therapies. The introduction of imatinib established proof of principle for oncogene-targeted therapy, leading to sustained survival improvements. Second- and third-generation TKIs further enhanced response depth and addressed resistance, including the T315I mutation. More recently, the development of the allosteric inhibitor asciminib introduced a novel mechanism of action and expanded therapeutic options for pretreated patients. Furthermore, the achievement of deep molecular responses has enabled TFR in approximately 40–60% of selected patients, redefining treatment goals toward functional cure. Emerging agents, including next-generation ATP-competitive and allosteric inhibitors, are showing promising activity in resistant disease and may further improve outcomes. Thus, CML represents a unique model of translational oncology, demonstrating how mechanistic insight can drive therapeutic innovation. Future strategies will focus on increasing TFR rates, overcoming resistance, targeting leukemic stem cells, and improving global access to therapy and monitoring, with the ultimate aim of achieving functional cure in the majority of patients. Full article

Background: Peripheral neuropathy is a common and clinically heterogeneous neurological condition caused by metabolic, inflammatory, toxic, or traumatic factors and is associated with sensory deficits, neuropathic pain, motor impairment, and reduced functional capacity. Management remains challenging and often requires multimodal therapeutic approaches, as pharmacological monotherapy frequently provides incomplete symptom control. Objective: This narrative review explores the conceptual rationale for combining galantamine with iontophoresis and Black Sea brine-based therapy as a potential multimodal strategy for peripheral neuropathy management. Main Findings: Galantamine, a reversible acetylcholinesterase inhibitor and positive allosteric modulator of nicotinic acetylcholine receptors, has demonstrated neuroprotective, neuromodulatory, and anti-inflammatory properties in experimental settings. Iontophoresis may provide a non-invasive method for targeted local drug delivery while reducing systemic exposure. Black Sea brine, widely used in Bulgarian balneological and rehabilitation practice, has been associated with improved circulation, pain reduction, and neuromuscular support. The reviewed evidence suggests biologically plausible complementary mechanisms; however, no direct clinical studies evaluating the combined intervention were identified. Limitations: Current evidence is indirect and derived from separate investigations of galantamine, iontophoresis, and brine-based therapy, as well as heterogeneous historical and regional sources. Therefore, the proposed combination should be considered hypothesis-generating rather than evidence-established. Conclusions: The combination of galantamine, iontophoresis, and Black Sea brine represents a potentially interesting multimodal concept for peripheral neuropathy rehabilitation. Well-designed preclinical and clinical studies are required to determine safety, feasibility, optimal treatment parameters, and therapeutic efficacy. Full article