Abstract: Oleanolic acid (OA) is a hydrophobic pentacyclic triterpene widely distributed in the plant kingdom and characterized by broad biological activity, including antioxidant, anti-inflammatory, neuroprotective, renoprotective, and anticancer effects. Increasing evidence suggests, however, that many of these actions are better explained not by single molecular targets, but by OA-dependent modulation of an integrated organelle stress network involving mitochondria, the endoplasmic reticulum (ER), autophagy, mitophagy, and apoptosis. This review critically analyzes the available evidence on the effects of OA on the mitochondria–ER–autophagy–apoptosis axis, with particular emphasis on mechanisms governing the transition between cellular adaptation and cell death. The available literature indicates that, in non-cancer models, OA most commonly lowers reactive oxygen species (ROS), stabilizes mitochondrial function, attenuates the ER stress signature, and promotes adaptive autophagy and mitophagy. In contrast, in many cancer models, OA may enhance mitochondrial dysfunction, lower the threshold for mitochondrial apoptosis, and induce autophagy that can be either protective or cytotoxic depending on the biological context. Overall, the current evidence supports a model in which OA acts as a context-dependent modulator of the organelle stress threshold rather than as a uniformly cytoprotective or uniformly proapoptotic compound. At the same time, the literature remains heterogeneous with respect to models, doses, exposure times, and markers used, while poor aqueous solubility and limited bioavailability continue to constrain translation. Future studies should therefore integrate analyses of mitochondria, ER, mitochondria–ER contact sites (MERCS), autophagy, apoptosis, pharmacokinetics, formulation, and safety in order to define the true potential of OA as a modulator of biological stress.

Abstract: Belonging to the Euphorbiaceae family, Jatropha genus is a promising source for discovering of antitumor compounds. Jatropha ribifolia is a traditionally used species in folk medicine in the semi-arid region of Brazil with a few chemical and pharmacological reports. Based on that, the aim of the current work is to isolate, structurally characterize, and assess the cytotoxic activity of isolated compounds through in vitro and in silico analyses. To achieve these main goals, the underground parts were dried, extracted and purified using classical and instrumental chromatographic techniques, leading to the isolation of 16 compounds. Altogether with HR-ESI-MS, IR, one- and two-dimensional NMR experiments, eight previously unreported diterpenes, named ribifolones A-H, along eight known compounds, were obtained and are herein described. Regarding their activity against melanoma (SK-MEL-28) and colorectal cancer (HCT-116) cell lines, jatrophone was the most potent with IC50 of 6.19 µM and 10.09 µM followed by ribifolone that exhibited a moderate cytotoxicity with IC₅₀ values of 50.71 µM and 33.39 µM, respectively. Network pharmacology analysis suggests the involvement of PI3K-AKT-mTor pathway in the activity of both compounds, meanwhile molecular docking and dynamics simulations demonstrate the main interactions with key proteins in the pathway and highlighted the estrogen receptor beta (ERβ) as putative target. This work opens new perspectives for the discovery of bioactive compounds found in Euphorbiaceae species, especially from those occurring in Caatinga.

Abstract: Alzheimer’s disease (AD) represents a escalating global neuropharmacological crisis, with prevalence in high-growth demographic regions such as India projected to exceed 14 million by 2040. This study addresses the urgent need for high-potency, dual-site acetylcholinesterase (AChE) inhibitors through an integrated computational pipeline. Background: We address the failure of mono-target paradigms by designing scaffolds capable of simultaneously anchoring the Catalytic Active Site (CAS) and the Peripheral Anionic Site (PAS). Methods: A robust GA-MLR QSAR model was developed from 115 quinoline analogues using 11,135 descriptors. Lead candidates were prioritized via blind molecular docking (7XN1) and 100-ns molecular dynamics (MD) simulations. Results: The five-descriptor model (R2 = 0.7569, QLOO2 = 0.7244) was validated by an external set of 8 experimental compounds (Rext2 = 0.8620). Lead Compound 19 emerged as a superior candidate (ΔG = -11.1 kcal/mol), exhibiting a stable MD trajectory (PL-RMSD ≈ 2.4 Å) and preserving essential Gly121-His447 catalytic anti-correlations. Conclusions: This study provides a statistically validated scaffold and mechanistic foundation for future biomimetic chromatography validation, advancing the high-throughput screening of neuroprotective agents on a global scale.

Abstract: Human African Trypanosomiasis (HAT) and malaria are serious infectious diseases endemic in tropical regions, caused by protozoan parasites, and necessitating an urgent development of new antiprotozoal drugs. As part of our ongoing search for new antiprotozoal steroidal alkaloids from plants, we investigated the methanolic stem bark extract of Holarrhena pubescens (Apocynaceae). H. pubescens is a tropical tree that some Kenyan coastal communities have long used to treat various ailments, including fever and stomach pain. The crude extract, alkaloid fraction, and 16 subfractions acquired through centrifugal partition chromatography (CPC) displayed promising in vitro antiprotozoal activity against Trypanosoma brucei rhodesiense (Tbr) and Plasmodium falciparum (Pf). Partial least squares (PLS) regression modelling of UHPLC/+ESI QqTOF-MS data and antiprotozoal activity data of the crude extract and its fractions was performed to predict compounds that may be responsible for the observed antiplasmodial activity. Chromatographic separation of the alkaloid fraction afforded one new steroidal alkaloid (5), along with 18 known compounds (1, 2, 4, 6–20), and one artifact (3) that was presumably formed during the acid-base extraction process. The structural characterization of the isolated compounds was accomplished using UHPLC/+ESI-QqTOF-MS/MS and NMR spectroscopy. The isolated compounds were tested for their in vitro antiprotozoal properties against the two aforementioned pathogens, as well as for their cytotoxicity against mammalian cells (L6 cell line). Compounds 2 and 16 (IC₅₀ = 0.2 μmol/L) demonstrated the highest antitrypanosomal activity, with compound 2 showing the highest selectivity (SI = 127). The new compound 5 exhibited the strongest antiplasmodial activity and selectivity against Pf (IC₅₀ = 0.7 μmol/L, SI = 43). Our findings provide further promising antiprotozoal leads for HAT and Malaria.

Abstract:

Many cannabinoids are derived from Cannabis and exhibit a diverse range of pharmacological properties. Predictions of bioactivities of these compounds were conducted by molecular docking computation on two transient receptor potential (TRP) receptors (TRPV1 and TRPC5) found on human glioma (U-87 MG) cells. These predictions were experimentally confirmed by monitoring changes in intracellular calcium concentration in U-87 MG cells treated with cannabinol (CBN), cannabichromene (CBC), and cannabicyclol (CBL), as measured using a fluorescence microplate reader. The results indicate that CBN and CBC are bioactive, whereas CBL exhibits minimal activity. These findings are consistent with predictions obtained from molecular docking computation based on AutoDock Vina.

Abstract: The c-Jun N-terminal kinase (JNK) signaling cascade is a central regulator of cellular stress responses and a validated therapeutic target for diverse pathologies, including neurodegeneration, fibrosis, and cancer. However, the development of direct JNK inhibitors has been impeded by challenges regarding isoform selectivity and on-target toxicity. Consequently, medicinal chemistry efforts have shifted upstream to the "gatekeepers" of the pathway: the dual-specificity kinases MKK4 and MKK7. This review provides a comprehensive analysis of the structural biology, physiological functions, and pharmacological inhibition of these critical signaling nodes. We highlight the non-redundant therapeutic potential of these kinases, contrasting the role of MKK4 inhibition in unlocking liver regeneration with the utility of MKK7 inhibition in suppressing fibrosis and chronic inflammation. A systematic overview of small-molecule inhibitors is presented. For MKK4, we discuss the evolution from early promiscuous chemotypes to the first-in-class clinical candidate HRX215, which has demonstrated safety and efficacy in promoting hepatocyte proliferation. For MKK7, we examine the design of covalent inhibitors exploiting the unique hinge-region cysteine (Cys218), as well as emerging modalities such as lysine-targeting binders and peptide inhibitors disrupting protein-protein interactions. Finally, we discuss remaining challenges and future opportunities, including the development of dual inhibitors and proteolysis-targeting chimeras (PROTACs), to fully exploit the therapeutic value of the MKK4/7-JNK axis.

Abstract: Acmella oleracea (L.) R. K. Jansen (Asteraceae), commonly known as the "toothache plant" or "jambu," is a significant medicinal plant that has been traditionally used in Brazil and other tropical and subtropical regions for relieving dental pain, as an anti-inflammatory agent, and as a culinary spice. Due to its versatile utility, this plant has been extensively studied in modern medicine and pharmacy for its diverse pharmacological properties, including anesthetic, analgesic, anti-inflammatory, antioxidant, and antimicrobial activities. Analytical research on the chemical compositions responsible for these activities has led to the identification of approximately 120 secondary metabolites. These findings provide scientific validation for its traditional uses and have spurred research into the development of ingredients for functional foods and cosmetics. This review incorporates the latest research findings, focusing on biological activities and compounds that have been practically isolated or can be isolated based on quantitative experimental data, to serve as a practical reference for industrial development. Furthermore, factors influencing the content of alkylamides and phenolic compounds, two major bioactive groups, are summarized to support material development. Ultimately, this review aims to provide a clearer understanding of the plant’s utility and contribute to the development of products that enhance human health.

Abstract: The human metapneumovirus (HMPV) Fusion (F) glycoprotein is a high-priority target for "fusion-locking" agents that stabilize its metastable prefusion state. While monomeric catechins like EGCG are known antivirals, the molecular basis for the superior activity of structurally complex dimeric catechins remains poorly understood. We employed an advanced biophysical workflow, integrating 100 ns all-atom Molecular Dynamics (MD), Free Energy Landscape (FEL) analysis, and MM/GBSA thermodynamic integration to decode the Structure-Dynamics Relationship (SDR) of 210 Camellia sinensis (Green tea) phytochemicals. The results reveal a "Galloylation-Driven Anchoring" mechanism: the galloyl moiety of prodelphinidin A2 3′-gallate provides critical electrostatic complementarity to the Asp325-Asp336 acidic ridge. FEL analysis quantitatively demonstrates that this anchoring traps the F protein in a deep, kinetically stabilized global minimum (ΔG = 9.357 kJ/mol), effectively raising the energy barrier for the fusogenic conformational shift. This study provides a rigorous thermodynamic proof-of-concept for the use of dimeric natural scaffolds as precision fusion-locking agents, offering a roadmap for experimental biophysical validation.

Abstract: Photodynamic therapy (PDT) offers a promising complementary strategy for the treatment of glioblastoma multiforme (GBM); however, achieving selective activation in tumor tissue and maintaining efficacy under hypoxic conditions remain significant limitations. In this study, we present the synthesis and functional evaluation of Gal-SiX, an enzymatically activatable Si-xanthene photosensitizer designed to address these challenges. Prepared through an improved 10-step synthetic route, Gal-SiX displays a clear turn-on fluorescence and absorbance response upon β-galactosidase activation and generates reactive oxygen species efficiently in aqueous media. Mechanistic studies revealed that Gal-SiX enables both Type I and Type II PDT pathways, an advantageous feature for GBM, where oxygen availability is restricted. In vitro assays conducted on U87MG glioblastoma cells and L929 healthy fibroblasts demonstrated meaningful selectivity, with IC50 values of 3.30 μM and 7.19 μM, respectively. Gal-SiX also showed minimal dark toxicity (>80 μM) and potent light-induced cytotoxicity, yielding a phototoxicity index of 24.8 in glioblastoma cells. Confocal imaging and MTT assays consistently demonstrated its activation and PDT efficacy. Overall, this work introduces the first activatable Si-xanthene–based PDT agent for glioblastoma and provides the first evidence that the Si-xanthene scaffold can support dual Type I/II phototoxicity. These results underscore Gal-SiX’s potential as a selective PDT platform for addressing the unique constraints of GBM biology.

Abstract: Green cosmetics are mainly based on plant-derived ingredients, using sustainable bio-technological tools for their preparation. The present research aimed to investigate the Usnea barbata extract in Jojoba oil (JO) enriched with 10% Vitamin E and 5% Pepper-mint oil (PEO), as a potential natural product for skin applications. Materials and Methods: The U. barbata extract (UBPJO) was obtained through cold maceration. Phytochemical screening was performed using Gas Chromatography/Mass Spectrometry (GC-MS), Folin Ciocalteu method, and Graphite-Furnace Atomic Absorption Spectro-photometry. The physicochemical properties were evaluated by Fourier Transform Infrared Spectroscopy and Atomic Force Microscopy. Then, rheological characteristics and oxidation stability (measuring the time required to reach the oxidation starting point, IP) of both oil samples (PJO and UBPJO), were investigated. Results: Total phenolic content in UBPJO was 2.5 times higher than in PJO (p < 0.05), while heavy metal levels (As and Pb) were slightly higher (p > 0.05). UBPJO has higher shear stress, viscosity, and spreadability than PJO, but without significant differences (p > 0.05). Finally, IP measurements indicated appreciable oxidative stability (UBPJO vs. PJO: 153.02 h vs 137.35 h, p > 0.05). Conclusions: The phytochemical composition and physicochemical properties support the inclusion of UBPJO in various skin-protective formulations.

Abstract: In 2025, the U.S. Food and Drug Administration (FDA) approved 44 new drugs, reflecting a slight decrease compared to previous years but maintaining the overall trends in pharmaceutical inno-vation. Biologics accounted for 25% of approvals, including eight monoclonal antibodies (mAbs), two antibody–drug conjugates (ADCs), and one fusion protein, with cancer remaining the primary therapeutic focus. TIDES, comprising three oligonucleotides and one peptide, continued to con-solidate their presence in the market, with the three oligonucleotides featuring N-acetylgalactosamine (GalNAc) for liver-targeted delivery. Small molecules dominate the re-mainder, with a high prevalence of N-aromatic moieties and fluorine atoms present in most of the molecules.. Peptide manufacturing and sustainability concerns, including PFAS usage, remain key challenges. Despite these advances, the high cost of innovative therapies limits access, particularly in low- and middle-income countries. This report provides a structural and chemical analysis of the newly approved drugs, highlighting trends in molecular design, therapeutic areas, and technolog-ical innovations shaping modern drug discovery.

Abstract: Neuroblastoma, a leading cause of pediatric cancer mortality, requires efficient and selective therapeutics. We synthesized and characterized a series of sulfonated 5-piperazine-containing 1,3-oxazole-4-carbonitrile derivatives via sulfonylation protocols and evaluated their in vitro cytotoxicity against human hepatocellular carcinoma (HepG2, Huh7), breast (MCF7, MDA-MB-231), cervical (HeLa), melanoma (M21), and two neuroblastoma (Kelly, SHSY5Y) cell lines, alongside non-malignant HEK293 cells. Three compounds (7a, 7b, and 8aa) demonstrated efficient and selective cytotoxicity toward MYCN-amplified (Kelly) and MYCN-non-amplified (SHSY5Y) neuroblastoma cells, with 7b being the most active (IC₅₀ = 1.9 µM in Kelly cells) and showing minimal toxicity in HEK293 (IC₅₀ > 10 µM). In Kelly cells, 7b matched doxorubicin while showing lower off-target toxicity and low-micromolar activity in solid tumors. The hit compounds were shown to be not readily biodegradable under OECD 301D test. In silico docking revealed high-affinity binding to the ATP-binding site of Aurora A kinase in the Aurora A/N-MYC complex (ΔG = –10.8 to –10.9 kcal/mol). Compound 7b exhibited broad cytotoxicity across all tested cancers (IC₅₀ = 1.5–4.0 µM). ADMET profiling of 7a, 7b, and 8aa indicated favorable pharmacokinetic and toxicity parameters compared with doxorubicin. These findings identify sulfonylated oxazole-4-carbonitriles as neuroblastoma therapeutics with potential use against proliferating malignancies.

Abstract: Marine triterpenoid saponins are structurally diverse metabolites with high pharmacological and nutraceutical potential, yet their characterization remains challenging due to extensive isomerism, aggregation phenomena, and the frequent co-extraction of lipids and other matrix components. In this work, we combine ATR–FTIR and high-resolution LC–MS to investigate the spectral and chromatographic behaviour of Cucumaria frondosa extracts and butanol-enriched fractions. FTIR spectra reveal a strong aliphatic signature, N–H-related features, and ester carbonyl bands consistent with the presence of co-extracted lipids and nitrogen-containing species such as ceramides or sphingolipids. LC–MS analysis of preparative fractions shows recurrent saponin-like ions— most prominently a feature at m/z ≈ 1347—reappearing across chromatographically distinct fractions, often accompanied by lipid-like ions in the 600–900 m/z range. These observations indicate that closely associated lipidic species can modulate the apparent chromatographic behaviour of saponin-containing fractions. Comparison with the Marine Animal Saponin Database (MASD v1.0) highlights both its value and its current MS1-centric limitations, including the lack of diagnostic MS/MS spectra and occasional inconsistencies between reported formulas and listed molecular weights. These findings underscore the need for integrated, multi-spectroscopic workflows and standardised spectral libraries to support confident annotation of marine saponins. Rather than proposing new structures, this study validates an analytical workflow that bridges early-stage MS screening with preparative fractionation and orthogonal spectroscopic assessment, offering a methodological reference to minimise misidentification and to guide future structural and biological investigations of marine triterpenoid saponins.

Abstract: Osteoarthritis (OA) is a prevalent chronic pain syndrome and a leading cause of disability worldwide, characterized by progressive deterioration of articular cartilage. This degradation leads to pain, swelling, inflammation, and eventual stiffness as the cartilage wears down, causing bone-on-bone friction. Current medical treatments primarily aim at pain relief; however, many interventions, especially invasive or surgical ones, carry risks of adverse outcomes. Consequently, intra-articular (IA) therapy, particularly hyaluronic acid (HA) injections, is widely adopted as a conservative treatment option. HA plays a crucial role in maintaining joint homeostasis by supporting proteoglycan synthesis and scaffolding, restoring optimal HA concentrations in synovial fluid, and providing chondroprotective and anti-inflammatory effects. In recent years, hydrogels composed of natural and synthetic materials have emerged as promising candidates for OA treatment. Our research focuses on the biosynthesis and characterization of novel hydrogel composites combining short peptide hydrogelators with aminated graphene oxide (a-GO) nanosheets functionalized with HA (a-GO-HA@Hgel). These a-GO-HA@Hgel nanocomposites are designed to facilitate the controlled release of HA into the extracellular matrix, aiming to promote cartilage regeneration and mitigate inflammation. The strategy is to exploit the oxygen-containing functional groups of GO nanosheets to enable covalent coupling or physical adsorption of HA molecules through various chemical approaches. The resulting a-GO-HA are incorporated within hydrogel matrices to achieve sustained and controlled HA release. We study the influence of a-GO-HA on the native hydrogel structure and its viscoelastic properties, which are critical for mimicking the mechanical environment of native cartilage tissue. Through this multidisciplinary approach combining advanced materials science and cellular biology, this work aims to develop innovative nanocomposite hydrogels capable of delivering HA in a controlled manner, enhancing cartilage repair and providing a potential therapeutic strategy for OA management.

Abstract: We herein demonstrate that the thiosemicarbazone (TSC) ligand N'-(di(pyridin-2-yl)methylene)-4-(thiazol-2-yl)piperazine-1-carbothiohydrazide (HL) can coordinate with Ga3+ to give the cationic complex of [Ga(L)2]NO3 featuring an the octahedral Ga(III) center. [Ga(L)2]NO3 may undergo metathesis with both Fe2+ and Fe3+ and result in the formation of respective Fe2+- and Fe3+ complex. Meanwhile, [Ga(L)2]NO3 is also susceptible to anion exchange with solidum hyaluronate (NaA) to produce the nanoformulation of [Ga(L)2]A with boosted aqueous solubility and cell targetability. [Ga(L)2]A demonstrated remarkable in vitro cytotoxicity against NCI-H82 and A549 (lung cancer), as well as KYSE-510 and Te-1 (esophageal cancer) cell lines, featuring half maximal inhibitory concentration (IC50) values in the range of 0.102 − 2.616 μmol L−1. This work highlights the potential of using non-toxic and biocompatible Ga3+ as the central ion to prepare TSC-based nanomedicines for combating cancer.

Abstract: Expanding the chemical diversity of DNA-encoded libraries (DELs) is crucial for identifying binders to emerging drug targets using DEL technology. In the present study, as part of our ongoing efforts to develop on-DNA diazide platforms (D-DAPs)—platform molecules possessing both aromatic and aliphatic azide groups on a single core reactive scaffold—we have designed and synthesized a new compact diazide platform, designated as a compact D-DAP (C-D-DAP). This molecule is based on a low-molecular-weight reactive scaffold, 3-azido-5-(azidomethyl)benzoic acid, to facilitate small-molecule drug discovery targeting enzymes and G protein-coupled receptors (GPCRs). Furthermore, we established two distinct stepwise warhead construction strategies that exploit the chemoselective transformations of the azide groups in the C-D-DAP, which exhibit different reactivities. In addition, four virtual DELs were generated based on stepwise warhead elaboration from the C-D-DAP scaffold. Comparative chemical diversity analysis against bioactive compounds from ChEMBL revealed that these virtual libraries populate structural regions that are sparsely represented among known molecules. Each virtual library also occupies a distinct region of structural space relative to the others and displays intermediate quantitative estimate of drug-likeness (QED) values.

Abstract: Hypertension is a leading global health burden, with dihydropyridine calcium channel blockers (DHP CCBs) serving as a primary therapeutic class. However, the molecular and pharmacokinetic determinants underlying their variable clinical efficacy remain incom-pletely understood. This in silico study investigated the structural and ADME basis for the differential activity of five DHP drugs (amlodipine, nifedipine, isradipine, nicardipine, nisoldipine) targeting the L-type calcium channel CaV1.2. Molecular docking (Glide-XP), MM-GBSA binding free energy calculations using the human CaV1.2 structure (PDB: 8WE8), and ADME predictions (QikProp, CYP3A4 site of metabolism) were integrated. Results identified a conserved hydrogen bond with residue SER1132 (bond length range: 1.931–2.094 Å) as a key binding anchor. The Coulombic interaction energy (range: -74174.2 to -74202.3 kcal/mol) showed a strong inverse correlation with experimental IC₅₀ (0.013–0.194 µM), establishing it as a primary affinity determinant. Pharmacokinetically, predicted human serum albumin binding (QPlogKhsa: 0.237–0.770) directly correlated with IC₅₀, and metabolic vulnerability to CYP3A4 varied notably among the drugs. These findings demonstrate that the differential potency of DHP CCBs arises from a combination of target engagement strength, governed by electrostatic interactions and a conserved SER1132 anchor, and key ADME properties, providing a computational framework for ra-tional antihypertensive drug design.

Abstract: The development of Multi-Target-Directed Ligands (MTDLs) offers a compelling therapeutic strategy for multifactorial diseases like cancer and Alzheimer's disease (AD), which share pathological pathways, notably microtubule abnormalities. This study introduces and validates a state-of-the-art computational pipeline, the QSAR-MD-DCCM workflow, designed to accelerate the discovery of dual-acting agents targeting tubulin polymerization and acetylcholinesterase (AChE). Two highly predictive QSAR models (R2 &gt; 0.83), built upon the trimethoxyphenyl scaffold, guided the rational design of 16 novel compounds. Subsequent ADMET screening identified compounds 15 and 16 as optimal leads, demonstrating excellent physicochemical properties and CNS penetrability. Molecular docking and rigorous 100 ns Molecular Dynamics (MD) simulations confirmed strong, persistent binding to both targets (PDB ID: 4O2B for tubulin; 1EVE for AChE), with the compounds showing complementary, target-differentiated potency. Subsequent MM-GBSA/MM-PBSA binding free energy calculations provided the essential energetic validation, confirming highly favorable binding for both leads. Crucially, Dynamic Cross-Correlation Map (DCCM) analysis provided novel mechanistic insights into the functional allosteric coupling of residues upon ligand binding, reinforcing the stability and distinct dynamic modes of action for both compounds. This integrated methodological approach successfully delivered two highly validated virtual MTDL candidates, establishing a robust and predictive platform for accelerating dual-target drug discovery.

Abstract:

Equinin B (GQCQRKCLGHCSKKCPKHPQCRKRCIRRCFGYCL), a marine peptide from Actinia equina exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria. To identify a smaller active region, the peptide was cleaved into three fragments: GQCQRKCLGHCS (EB-1), KKCPKHPQCRK (EB-2) and RCIRRCFGYCL (EB-3). Only the 11-residue C-terminal fragment showed selective activity against Gram-positive bacteria, including Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus hirae, while remaining inactive against Escherichia coli. Peptide modifications, achieved by replacing cysteine residues with arginine, generally did not enhance activity, but in the C-terminal fragment they reduced hemolytic activity and increased bacterial specificity. Membrane depolarization assays confirmed that the unmodified fragment strongly disrupts bacterial membranes, whereas the modified variant showed minimal depolarization, highlighting its markedly reduced membrane-disruptive potential. In silico modelling revealed that the unmodified fragment (EB-3) can adopt multiple membrane-disruption modes, from transient shallow pores to carpet-like mechanisms, while the cysteine-to-arginine variant interacts mainly via partial insertion anchored by arginine residues. Phenylalanine appears to interact with the membrane, and reducing hydrophobicity by its removal abolished antibacterial activity. These findings highlight the 11-residue C-terminal fragment as a tunable, membrane-targeting motif with mechanistic novelty, offering a blueprint for developing safer, selective antimicrobial peptides with reduced cytotoxicity.

Abstract: Background/Objectives: Escalating resistance to existing antifungal compounds necessitates development of novel bioactive molecules with innovative mechanisms. Paclobutrazol, a triazole-containing plant growth regulator with modest antifungal activity, presents a structurally versatile scaffold amenable to derivatization. This study investigated whether strategic structural modifications could enhance antifungal potency and reveal broader therapeutic applications through integrated computational approaches. Methods: Twenty-six novel paclobutrazol derivatives were synthesized via etherification or esterification and characterized through NMR spectroscopy (¹H, ¹³C, ¹⁹F), IR spectroscopy, and mass spectrometry. Computational assessment employed CropCSM for toxicity prediction, pdCSM-GPCR for G protein-coupled receptor affinity estimation, MolPredictX for pathogenic target activity probability, CB-Dock2 for molecular docking, and pkCSM for ADME properties and drug-likeness evaluation. Results: Compound 26, featuring naphthyl substitution, demonstrated strong binding to sterol 14-alpha demethylase (-10.8 kcal/mol), calcitonin gene-related peptide type 1 receptor (-11.1 kcal/mol), extracellular calcium-sensing receptor (-10.9 kcal/mol), and metabotropic glutamate receptor 4 (-10.4 kcal/mol), with CGRP1R affinity comparable to approved antagonist rimegepant (-11.3 kcal/mol). Compounds 18 and 19, containing nitro groups, were the only substances predicted to exhibit AMES toxicity. Multiple derivatives showed activity against fungal, bacterial, parasitic, and viral targets. Compounds 20-22 displayed favorable drug-like properties with balanced physicochemical parameters. Conclusions: This work establishes paclobutrazol as a viable scaffold for therapeutic development beyond traditional antifungal applications. The analysis suggests potential utility in migraine and pain management through CGRP receptor modulation, calcium homeostasis disorders via calcium-sensing receptor targeting, neuroendocrine conditions through somatostatin receptor type 5, and inflammatory diseases via prostaglandin D2 receptor 2. These findings provide a framework for repurposing agricultural compounds in drug discovery, though further experimental validation is required.