Acta Pharmaceutica Sinica B

Despite the development of vaccines and antivirals, coronavirus disease 2019 (COVID-19) continues to affect populations worldwide. Given the high mutation rate of the SARS-CoV-2 virus and reports of drug resistance, there is a continued need for new therapeutic options. SARS-CoV-2 main protease (Mpro) is essential for viral replication and is a conserved target among coronaviruses. Most known Mpro inhibitors target the active site, although allosteric sites have already been identified. In this study, we conducted a virtual screening of 2,060 compounds targeting an allosteric site of SARS-CoV-2 Mpro. From this screen, 41 computational hits and analogs were selected and evaluated using biochemical assays against SARS-CoV-2 Mpro. Among them, compound 25, a semicarbazone, demonstrated a half-maximal inhibitory concentration (IC50) of 99 M. Additionally, two thiosemicarbazone analogs (compounds 50 and 51) inhibited SARS-CoV-2 Mpro with IC50 values of 61 M and 70 M. Biochemical assays suggest that these compounds act as noncovalent competitive inhibitors of SARS-CoV-2 Mpro. Molecular dynamics simulations revealed that compound 25 is unstable at the allosteric site of SARS-CoV-2 Mpro but forms stable and favorable interactions at the active site, supporting its potential as a competitive inhibitor, a finding subsequently confirmed by biochemical assays. Our structure-based computational and biochemical approach identified semicarbazone and thiosemicarbazone scaffolds as promising candidates for the development of reversible SARS-CoV-2 Mpro inhibitors.

The chikungunya virus (CHIKV) outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV. A CHIKV macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer (FRET)-based assay, and its inhibitory activity was validated through multiple orthogonal assays. Compound 1 has a dual thiobarbiturate-indole scaffold and exhibits an IC50 of 8.9 {micro}M. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Notably, the inhibitor shows high selectivity for the CHIKV macrodomain over a panel of human and viral ADP-ribosyl binding and hydrolyzing proteins. Structure-activity relationship studies and medicinal chemistry efforts provide a promising starting point for further hit optimization.

The enzymatic function of ABHD6 on insulin secretion and insulin resistance is well documented. However, its non-enzymatic function, especially its effects on selective hepatic insulin resistance and metabolic dysfunction-associated steatotic liver disease (MASLD) is completely unexplored. ABHD6 is elevated under conditions of diet-induced obesity and aging. To define the role of ABHD6 in liver physiology, we generated liver-specific ABHD6 knockout mice, as well as liver specific overexpression of native and enzymatic inactive mutant ABHD6 mouse models. We demonstrated that ABHD6 is an unidentified regulator of selective hepatic insulin resistance and contributes to MASLD and liver fibrosis. Furthermore, we found that non-enzymatic ABHD6, rather than its enzymatic form, contributes to this regulation. Mechanistically, we found that ABHD6 translocated into the nucleus and interacted with Akt/FoxO1 axis to regulate its function. In addition, knockdown of FoxO1 in primary hepatocytes or overexpression of constitutively active mutant FoxO1 by AAV approach could completely abolish the effects of ABHD6 on glucose tolerance and gluconeogenesis. Our study reveals an entirely different mechanism underlying selective hepatic insulin resistance that involves a previously unknown non-enzymatic function of ABHD6. This study opens an avenue for the development of a novel class of ABHD6 inhibitors to treat MASLD and liver fibrosis. HighlightsO_LIABHD6 expression in the liver is increased with obesity and aging. C_LIO_LIABHD6 manipulation affects selective hepatic insulin resistance, MASLD and liver fibrosis. C_LIO_LINon-enzymatic ABHD6 interacts with Akt/FoxO1 axis to regulate FoxO1 transcriptional activity. C_LIO_LIThe effects of ABHD6 on glucose tolerance and hepatic gluconeogenesis are completely dependent on FoxO1 activity. C_LI

Hyperglycemia is a hallmark of type-2 diabetes and a key pathogenic driver of diabetic complications. Cullin RING E3 ligases (CRLs) are multi-subunit E3 ubiquitin ligases that mediate cellular protein turnover. The activity of CRLs requires cullin neddylation, a post-translational modification that can be pharmacologically targeted with therapeutic potentials. By using hyperinsulinemic-euglycemic clamp analysis, we discover that pan neddylation inhibitor exerts both insulin sensitization effect in liver and muscle and insulinotropic effect in pancreatic {beta} cells. This dual action is mediated by Cullin 3 (Cul3), a member of the 7 canonical cullin family proteins. DI-1859, a selective Cul3 neddylation inhibitor, effectively protects against hyperglycemia in obese mice. DI-1859 enhances insulin signaling by preventing Cul3-mediated insulin receptor substrate degradation in liver and muscle cells. DI-1859 increases insulin secretion in a glucagon-like peptide-1-independent manner in mice and directly potentiates glucose-stimulated insulin secretion in INS-1 832/13 {beta} cells and human islets. Mechanistic studies reveal that DI-1859 does not promote glycolytic flux or bioenergetics function but potentiates glucose-stimulated insulin secretion via mechanisms involving RhoA activation and cytoskeleton remodeling in {beta} cells. This study shows that a single agent targeting Cul3 neddylation simultaneously promotes insulin sensitization and insulin secretion to attenuate hyperglycemia in mice. Article Highlightsa. Pan cullin neddylation inhibitors exhibit potent hypoglycemic effect. b. The target organs and mechanisms underlying the hypoglycemia effect of cullin pan neddylation inhibitors are incompletely understood. c. We found that inhibition of Cul3 leads to a dual insulin sensitization and insulinotropic effect. d. Selective inhibition of Cul3 neddylation is a feasible approach to lower hyperglycemia.

Sirtuins (SIRTs), which remove protein lysine acyl modifications, play crucial roles in diverse cellular processes, including metabolism, gene transcription, DNA damage repair, cell survival, and stress response. Several sirtuins are considered non-oncogene addiction of cancer cells and promising targets for anticancer drug development. High-throughput screening (HTS) methods for sirtuins are critical for the development of potent and isoform-selective sirtuin inhibitors, which are needed to validate the therapeutic potential. Herein, we designed and synthesized a fluorescent polarization (FP) tracer, KP-SC-1. Using this high-affinity tracer, we developed a robust, high-throughput FP competition assay for screening SIRT1-3 inhibitors. The assay was validated by testing known SIRT1-3 inhibitors. The assay can detect NAD+-independent SIRT1-3 inhibitors, as well as NAD+-dependent inhibitors, such as Ex-527 and TM. Finally, our assay showed satisfactory stability and outstanding performance in a pilot library screening. Compared to previous assays, the FP assay uses much less SIRT1-3 enzymes, a feature important for high-throughput library screening. We believe that the FP assay developed here will accelerate the discovery and development of SIRT1-3 inhibitors.

The glucagon-like peptide-1 receptor (GLP-1R) plays a central role in metabolic regulation and is a major therapeutic target for obesity and diabetes. Peptide agonists, like semaglutide, targeting the GLP-1R remain among the most effective regulators of glucose metabolism and appetite. Nonetheless, recent reports about weight regain have limited the effectiveness of GLP1R peptide agonists, sustaining the interest in expanding the chemical diversity of GLP-1R ligands through drug discovery strategies. However, the structural complexity and conformational plasticity of class B1 GPCRs make conventional single-method virtual screening approaches prone to bias and limited chemotype recovery. Using an integrated ligand- and structure-based virtual screening pipeline, explicitly combining complementary ligand-based descriptors, multi-fingerprint similarity, electrostatic similarity, pharmacophore modeling, and multi-conformation docking under a consensus-driven selection strategy, we were able to identify three chemically distinct classes of GLP-1R agonist candidates: GQB47810, a non-peptidic molecule; neuromedin C, a peptide, and 2,5-Pen-enkephalin (DPDPE), a small peptide. From all of them, DPDPE showed the greatest effectiveness, reaching values similar to those of GLP-1, although with lower potency. Further in vitro characterization confirmed that pen-enkephalin behaved as a full agonist and exhibited dual GLP-1R/GIPR agonistic activity. These findings establish a consensus-driven and transferable computational framework for chemotype-diverse agonist discovery at conformationally flexible GPCR targets, and revealed a pentapeptide with GLP-1-like efficacy as a promising lead for next-generation small peptide therapeutics.

Postprandial hyperglycemia is a major concern in type 2 diabetes, and inhibition of intestinal -glucosidases [maltase-glucoamylase (MGAM)] is an established method for controlling post-meal glucose excursions. In this study, we conducted an in-silico screening of phytochemicals from different well-known medicinal plants (Withania somnifera, Rauwolfia serpentina, Curcuma longa, and Camellia sinensis) against MGAM, using the clinically approved inhibitor miglitol as reference for docking protocol validation. Molecular docking revealed that miglitol binds to MGAM with a binding energy of -6.86kcal/mol and an RMSD of 1.04 (with co-crystal structure; PBD ID:3L4W); however, several phytochemicals exhibited binding affinities equal to or stronger than miglitol. Among these, Withanolide B (-9.25kcal/mol) and Withanone (-7.57kcal/mol) from Withania somnifera showed the highest predicted affinities, indicating robust engagement of the MGAM catalytic pocket. Rauwolfia serpentina alkaloids such as yohimbine (-8.50kcal/mol) and raubasine (-8.46kcal/mol) also displayed strong binding energies, whereas curcuminoids (curcumin -6.36kcal/mol; deoxycurcumin -6.35kcal/mol) and tea catechins (e.g., epicatechin gallate -6.85 kcal/mol) demonstrated moderate affinity. Interaction analysis showed that top-ranking compounds formed extensive hydrogen-bonding and hydrophobic interactions with key catalytic residues of MGAM, suggesting stable occupancy of the active site. In-silico ADME profiling predicted favorable gastrointestinal absorption for lead phytochemicals, supporting their potential for oral intestinal action. Collectively, these results identify plant-derived ligands with binding energies comparable to or exceeding that of miglitol, highlighting Withania somnifera withanolides as priority candidates for experimental validation in enzyme inhibition assays and glucose tolerance models, and providing a focused set of natural MGAM inhibitors for further translational investigation in postprandial glucose control.

Tuberculosis persists as a major global health threat, significantly exacerbated by the rise of drug-resistant strains. Cytochrome P450 of 124 family CYP124 from Mycobacterium tuberculosis (CYP124), implicated in host sterol metabolism and bacterial virulence, represents an emerging and promising therapeutic target. While its precise physiological role was previously debated, CYP124s confirmed ability to metabolize immunomodulatory host sterols underscores its pharmacological relevance. Utilizing surface plasmon resonance binding assays and UV-Vis spectral titration screening, we identified nine novel non-azole ligands for CYP124 from a library of 32 plant-derived and marine natural compounds. Among these hits, (25S)-5-cholestane-3{beta},4{beta},6,7,8,15{beta},16{beta},26-octaol (termed 15{beta}-octaol) and henricioside H2 (HD-4) induced characteristic difference spectra and formed long-lived inhibitory complexes with CYP124, exhibiting dissociation half-lives of 181 min and 65 min, respectively. However, their inhibitory potency was moderate, with IC50 values of approximately 86 M for 15{beta}-octaol and exceeding 100 M for HD-4. Complementary in silico molecular docking and analysis identified key conserved hydrophobic residues within the CYP124 active site crucial for ligand binding, suggesting a shared pharmacophore. Furthermore, structural similarity analysis revealed that 37 human endogenous metabolites, including known immunoregulatory sterols, bear resemblance to the identified CYP124 ligands. This finding points towards a potential sterol-mediated interplay at the host-pathogen interface. Collectively, these results provide a foundation for the future development of mechanism-based CYP124 inhibitors as therapeutics against multidrug-resistant tuberculosis.

TEA domain (TEAD) proteins bind co-activator Yes-associated protein (YAP) to regulate the expression of target genes of the Hippo pathway. The TEAD*YAP protein-protein interaction is not druggable, but TEADs possess a unique and deep palmitate pocket with a highly conserved cysteine located outside the TEAD*YAP protein-protein interaction interface. Here, we screen a fragment library of acrylamide electrophiles and identify a fragment that forms an adduct with the conserved palmitate pocket cysteine and inhibits TEAD4 binding to YAP. Synthesis of a focused set of derivatives and time- and concentration-dependent studies with four TEADs provide reaction rates and binding constants. Co-crystal structures of fragments bound to TEAD2 and TEAD3 reveal reaction at the conserved palmitate pocket cysteine but also at another less conserved cysteine located in the palmitate pocket of TEAD2 closer to the TEAD*YAP interface. These fragments provide a starting point for the development of allosteric acrylamide small-molecule covalent TEAD*YAP inhibitors.

Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation and contributes to diverse pathologies including ischemia-reperfusion injury and neurodegenerative disorders. Current ferroptosis inhibitors largely function as nonspecific radical-trapping antioxidants, limiting their clinical utility. We previously identified MESH1 as a key regulator of ferroptosis through its NADPH phosphatase activity. Here, we identify 4,5,6,7-tetrabromo-1H-benzotriazole (TBB) as a small molecule inhibitor of MESH1 with an IC50 value of 4.7 {+/-} 0.3 {micro}M. X-ray crystallography revealed the molecular determinants of TBB recognition which are corroborated through structure-activity relationships of TBB analogs. TBB protected multiple cell lines against ferroptosis in vitro, and this effect was mitigated by MESH1 knockdown, consistent with on-target activity. Furthermore, TBB reduced neuronal death in an ex vivo brain slice model of Alzheimers disease. Collectively, these findings establish TBB as a bona fide small-molecule MESH1 inhibitor that suppresses ferroptosis and establishes MESH1 as a promising therapeutic target. Graphical AbstractDepicting mechanism of TBB suppressing ferroptosis through the inhibition of MESH1. Figure Created with Biorender.com O_FIG O_LINKSMALLFIG WIDTH=131 HEIGHT=200 SRC="FIGDIR/small/706832v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1fd60e9org.highwire.dtl.DTLVardef@1e56518org.highwire.dtl.DTLVardef@15010c2org.highwire.dtl.DTLVardef@17c313a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Inflammation serves as a vital physiological process essential for preserving health and countering illness. Yet, persistent inflammation drives osteoclastogenesis and ongoing bone erosion in rheumatoid arthritis (RA), mainly via macrophage activation and overproduction of pro-inflammatory cytokines like TNF-, IL-1{beta}, and IL-6. Limitations of prolonged conventional treatments underscore the need for safer small-molecule inhibitors that address both inflammation and osteoclast formation. Chalcones, natural plant defense compounds, exhibit diverse pharmacological properties including anti-inflammatory, anticancer, antibacterial, antifungal, and antiparasitic actions, owing to their characteristic reactive , {beta}- unsaturated carbonyl moiety. This study assessed chalcone derivative 7a for its anti-inflammatory effects in vitro and in vivo, alongside its capacity to modulate osteoclast differentiation, offering the inaugural demonstration of its dual anti-inflammatory and anti-osteoclastogenic properties. In LPS-stimulated macrophages, 7a substantially curtailed nitric oxide production, curbed pro-inflammatory cytokines (TNF-, IL-1{beta}, IL-6), and concentration-dependently diminished iNOS and COX-2 expression while inhibiting reactive oxygen species levels. In vivo, oral 7a dosing potently alleviated carrageenan-evoked paw swelling and restored serum lactate dehydrogenase and C-reactive protein to normalcy. In LPS-exposed mice, it further lowered systemic cytokines and rectified dysregulated biomarkers such as LDH, ALP, ALT, AST, creatinine, and urea. Moreover, in RANKL-stimulated osteoclast cultures, 7a markedly suppressed osteoclastogenesis by downregulating pivotal markers like tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). Derivative 7a also enhances antioxidant defense--superoxide dismutase and catalase--via blockade of NF-{kappa}B and MAPK pathways. Overall, chalcone derivative 7a displays robust anti-inflammatory and anti-osteoclastogenic activity, positioning it as a compelling candidate for RA therapy.

The hexanucleotide repeat expansion (GGGGCC) in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein forms a complex with SMCR8 and WDR41 (CSW), which acts as a GTPase-activating protein (GAP) regulating small GTPases like ARF1 and RABs involved in intracellular trafficking. Although these findings implicated the ARF1 dysregulation in ALS/FTD and the critical need for validation of its inhibition as potential intervention, small molecules that target the interactions between CSW and ARF1 are lacking. In this study, we showed that the tyrosine-phosphorylated form (Tyr-782) of ASAP1, an ARF-GAP that inactivates ARF1, is increased in the motor cortex of both sporadic ALS and ALS with C9orf72 mutations. Overexpression of C9orf72 led to Golgi disorganization, partially mimicking the effects of ARF1 inhibitor brefeldin A on dispersion of Golgi apparatus. To identify a better strategy to enhance C9orf72 and ARF1 interactions, we applied rational design and virtual screening of a 40-million compound library of small molecules targeting the ARF1-CSW interface. Molecular docking, MM-GBSA binding energy, ADME/Tox profiles, and interaction analysis established MCULE-5095997944 as a top candidate for ARF1 modulation. MCULE-5095997944 demonstrated strong binding to ARF1 in the nanomolar range, reduced GTP-bound ARF1 levels upon ARF1 activation, and altered ARF1-dependent Golgi organization. These studies identified the first small molecule targeting ARF1-CSW interaction and further support ARF1 modulation as a potential therapeutic approach for ALS/FTD.

Pro-death Bax isoform Bax{Delta}2 forms protein aggregates in Alzheimers neurons, triggering stress granule formation and neuronal cell death. In seeking chemical ligands to prevent Bax{Delta}2 monomer aggregation, we performed in silico screening of FDA-approved drugs using computational docking. This screening identified a group of compounds that bind to the hydrophobic pocket of Bax{Delta}2. Subsequent wet-lab testing revealed that digoxin could block neuronal cell death at nanomolar concentrations (50 to 100 nM). Importantly, digoxins protective role is specific to Bax{Delta}2-induced cell death and is independent of its primary cardio-action on Na/K-ATPase. Further investigation suggests that digoxin does not significantly affect the formation of Bax{Delta}2 aggregates but may instead modulate Bax{Delta}2 protein levels. Although the therapeutic use of digoxin for Alzheimers disease is not feasible due to its narrow therapeutic window and toxicity, these findings open the door for chemical modification of digoxin, or development of similar compounds, to prevent Bax{Delta}2-mediated neuronal cell death in Alzheimers disease.

The extensive expression of STING in patients with non - small cell lung cancer (NSCLC) is closely associated with overall survival and other factors. Activation of the STING pathway can suppress NSCLC. However, the clinical translation of STING agonists remains hindered by challenges such as off-target effects, metabolic instability, and suboptimal pharmacokinetics. In this study, we engineered two oncolytic adenoviruses (OAds), OAd-HcGAS and OAd-McGAS, expressing human or murine cGAS, respectively, using an Ad5/3 chimeric adenovirus platform under regulation by the hTERT promoter to evaluatewhether OVs carrying the cGAS gene are capable of specifically activating the STING pathway within tumors and enhancing the anti - tumor efficacy of OVs both in vitro and in vivo.In vitro, OAd-HcGAS exhibited robust replication and potent cytolytic activity in tumor cells. It activated the STING-TBK1-IRF3 signaling axis, triggering a strong type I interferon (IFN-I) and pro-inflammatory cytokine response without compromising viral replication. In a murine Lewis lung carcinoma allograft model, intratumoral (i.t.) administration of OAd-McGAS led to substantial cGAS expression and consequential activation of the STING pathway. Moreover, the combination with anti-PD-L1 therapy resulted in tumor regression in over half of the cases. Notably, this armed oncolytic virus strategy enhanced the activation and infiltration of multiple immune cell populations. Collectively, these findings establish cGAS-expressing oncolytic adenoviruses as a novel and effective therapeutic strategy for lung cancer treatment. Graphical AbstractViral replication & Transgene expression & Cancer treatment

Antimicrobial resistance (AMR) in plant pathogenic bacteria poses a serious threat to global agriculture, necessitating the development of novel antibacterial agents targeting virulence mechanisms. This study presents an integrated bioinformatics-driven framework for the rational design and computational validation of Solres, a newly designed small molecule targeting key virulence proteins in phytopathogenic bacteria. Approximately 10,000 active compounds from PubChem BioAssay (AID: 588726) were analyzed using structural clustering and scaffold mining to identify conserved molecular motifs associated with antibacterial activity. Guided by high-frequency substructures, Solres was designed de novo and screened for structural novelty against PubChem, ChEMBL, and WIPO databases. Drug-likeness evaluation using Lipinskis Rule of Five confirmed favorable physicochemical properties. Molecular docking was performed against essential virulence regulators, including PhcA, PhcR, HrpB, PehA, and Egl from Ralstonia solanacearum and Xanthomonas spp., with active sites predicted using CaspFold. Docking analyses revealed strong binding affinities and stable interactions with key catalytic and regulatory residues. Complex stability and conformational integrity were further validated through molecular dynamics simulations. Quantum chemical descriptors, including HOMO-LUMO energy gap and dipole moment, supported the electronic suitability and reactivity profile of Solres. Collectively, this study demonstrates the effective integration of cheminformatics, structural bioinformatics, molecular simulations, and quantum chemical analyses for plant-focused antibacterial discovery. The compound Solres represents a promising lead candidate for mitigating bacterial wilt disease and provides a computational framework for future experimental validation and sustainable crop protection strategies against AMR-driven phytopathogens.

The G protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) is a key mediator of bile acid signaling, exerting its physiological effects through coupling with the stimulatory G protein (Gs). This interaction is essential for stabilizing the receptors active conformation and triggering downstream signaling. Among endogenous ligands, lithocholic acid (LCA) is the most potent natural agonist. However, the dynamic features underlying its binding and activation mechanisms remain poorly defined. In this study, we investigated the molecular basis of the interaction between LCA and GPBAR1, as well as the functional consequences of this interaction on receptor activation by integrating homology modelling, molecular docking, and molecular dynamics (MD) simulations. Our calculations reveal that LCA binding stabilizes the active state of GPBAR1, biasing the conformational ensemble of TM5 and TM6, as well as the main microswitches. These ligand-induced rearrangements enhance the coupling interface with the 5 helix of Gs and facilitate allosteric communication between the orthosteric and intracellular sites. Overall, our findings provide dynamic insight into how LCA modulates GPBAR1 activation and G protein engagement, highlighting its role as a molecular effector in bile acid signaling, and furnishing molecular detail relevant to ongoing efforts in GPBAR1-targeted compound development.

The RNA helicase DHX9 is essential for genomic stability, transcription, translation regulation and other RNA-related processes. DHX9 has emerged as a therapeutic target for cancer treatment as its expression levels are elevated in several different cancer types. Moreover, tumor cells exhibit a strong dependence on DHX9, making its inhibition an effective strategy for tumor regression. As RNA helicases are conserved enzymes, unique features need to be targeted to minimize side effects. Here, we identified an autoregulatory interface between the DHX9 helicase core and double-stranded RNA binding domain 2 (dsRBD2) ideal for a highly specific inhibition of DHX9. By targeting the proposed dsRBD2-core interface in DHX9, we aim to specifically inhibit DHX9 helicase activity by preventing dsRBD2 binding to the helicase core. We developed a protein binder design-based strategy targeting the dsRBD2-core interface of DHX9 by computationally designing novel dsRBDs. By binding this interface without engaging RNA, the dsRBD designs prevent dsRBD2-core interaction and inhibit DHX9 helicase activity. Our strategy of redesigning autoregulatory protein domains as inhibitors offers a computationally efficient alternative to larger-scale library generation and provides a flexible framework applicable to a wide range of therapeutic targets.

Rotavirus is a major cause of severe diarrheal disease in children under the age of five, with reduced vaccine effectiveness in low-resource settings causing substantial morbidity and mortality. In the absence of approved antiviral therapeutics, treatment is largely supportive, urging the need for targeted and precision-based interventions. VP4 protein plays an essential role in viral attachment, entry, and infectivity, making it a suitable target for targeted therapy. In this context, RNA interference is a specific method for inhibiting viral gene expression with its efficacy depending on sequence conservation, target accessibility, and compatibility with the RISC-loading machinery. In the present study, an integrative in silico approach was employed to design and evaluate siRNAs targeting conserved regions of the VP4 gene across six geographically diverse countries. Candidate siRNAs were screened using established design rules and regression-based scoring with off-target filtering. Three optimized siRNAs were further assessed through structural modeling, molecular docking, and molecular dynamics simulations to examine interactions with human Dicer, TRBP, and Argonaute-2. Comparative dynamic analyses identified one siRNA with enhanced structural compatibility, reduced conformational fluctuations, and stable interactions with RISC-loading proteins. These findings provide a rational computational basis for VP4-targeted siRNA development, facilitating experimental validation.

Brucellosis is a globally important zoonotic disease caused by Brucella melitensis, the most virulent and clinically significant species affecting both humans and livestock. Unlike many Gram-negative pathogens, B. melitensis, a facultative intracellular pathogen, lacks conventional virulence factors and instead relies on specialized systems such as the Type IV Secretion System (T4SS) for secretion of effector proteins. In this study, an integrated computational pipeline was implemented to identify, model, and assemble the T4SS components, encoded by virB operon, from the complete B. melitensis proteome. Template-based modeling strategies were employed to generate structures of T4SS subcomplexes, referencing crystallographic data from E. coli T4SS. Structural superposition with E. coli homologs revealed highly conserved architecture despite only 30-50% sequence identity. Stereochemical validation confirmed high model quality and favorable interactions among most VirB protein pairs. Membrane insertion analysis of the membrane-embedded assemblies further corroborated the spatial orientation of the modeled T4SS. Potential of T4SS as a drug target was explored by targeting dimeric interface of VirB11 ATPase to disrupt protein-protein interactions that could disarm the pathogen. Virtual screening of compounds from DrugBank database revealed compounds with docking score [≤] -7.0 kcal/mol that were screened based on ADMET properties, yielding three promising candidates - Ezetimibe (Drug Id: DB00973), Chlordiazepoxide (Drug Id: DB00475), and Alloin (Drug Id: DB15477). MM-GBSA analysis estimated favorable binding free energies for these compounds and molecular dynamics simulation for 200 ns further confirmed the protein-ligand interaction stability. Collectively, these findings provide new insights into the architecture of B. melitensis T4SS and identify three potential drug molecules targeting T4SS. This supports FDA - approved drug repurposing as an effective strategy for anti-virulence therapy against Brucellosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/706537v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@847c4borg.highwire.dtl.DTLVardef@1fc2551org.highwire.dtl.DTLVardef@f62a7corg.highwire.dtl.DTLVardef@15f3468_HPS_FORMAT_FIGEXP M_FIG C_FIG

Western-style diets promote chronic metabolic inflammation and dyslipidemia, yet safe interventions that restore immunometabolic homeostasis remain limited. Pyrroloquinoline quinone (PQQ) is a naturally occurring redox cofactor with antioxidant and metabolic regulatory properties, but its systemic effects in translational preclinical models are poorly defined. Here, we examined the impact of short-term PQQ supplementation in obese adult female olive baboons (Papio anubis) chronically fed a Western diet. Using a human-equivalent dose administered for 30 days, we found that PQQ supplementation significantly reduced circulating markers of systemic inflammation and cholesterol in Western-diet-fed animals, lowering circulating C-reactive protein, soluble CD163, and atherogenic lipoprotein fractions independent of changes in adiposity. Proteomic and pathway analyses of circulating proteins in plasma and serum revealed suppression of complement, thrombo-inflammatory, and extracellular matrix remodeling pathways, alongside enhanced lipoprotein assembly, remodeling, and clearance. Network analyses identified restoration of neurotrophic tyrosine kinase receptor 1 (NTRK1)- and forkhead box A2 (FOXA2)-regulated signaling as central features of the PQQ response, accompanied by inhibition of pro-fibrotic, xenobiotic, and inflammatory pathways, as well as predicted activation of liver X receptor (LXR)- and insulin growth factor (IGF)-associated metabolic programs. These findings demonstrate that PQQ rapidly reprograms systemic immunometabolic networks in a nonhuman primate model of diet-induced metabolic stress, highlighting FOXA2- and neurotrophin-associated pathways as novel targets of PQQs action.