Search Results (254)

Background: Pancreatic lipase (PL), the principal enzyme catalyzing the hydrolysis of dietary triacylglycerols in the intestinal lumen, is pivotal for efficient lipid absorption and plays a central role in metabolic homeostasis. Enhanced PL activity promotes excessive lipid assimilation and contributes to positive energy balance, key pathophysiological mechanisms underlying the escalating global prevalence of obesity—a complex, multifactorial condition strongly associated with metabolic disorders, including type 2 diabetes mellitus and cardiovascular disease. Inhibition of pancreatic lipase (PL) constitutes a well-established therapeutic approach for attenuating dietary lipid absorption and mitigating obesity. Methods: With the aim to identify putative PL inhibitors, a Structure-Based Virtual Screening (SBVS) of PhytoHub database naturally occurring derivatives was performed. A refined library of 10,404 phytochemicals was virtually screened against a crystal structure of pancreatic lipase. Candidates were filtered out based on binding affinity, Lipinski’s Rule of Five, and structural clustering, resulting in six lead compounds. Results: In vitro, enzymatic assays confirmed theoretical suggestions, highlighting Pinoresinol as the best PL inhibitor. Molecular dynamics simulations, performed to investigate the stability of protein–ligand complexes, revealed key interactions, such as persistent hydrogen bonding to catalytic residues. Conclusions: This integrative computational–experimental workflow highlighted new promising natural PL inhibitors, laying the foundation for future development of safe, plant-derived anti-obesity therapeutics. Full article

Isoquercitrin, a monoglucoside form of quercetin, exhibits superior antioxidant, anti-inflammatory, and cardiovascular protective effects in comparison to its precursor, rutin. However, its natural abundance is limited. This study aimed to increase the functional value of Diospyros lotus leaf extract through enzymatic conversion of rutin to isoquercitrin using α-l-rhamnosidase and to evaluate the changes in biological activities after conversion. A sugar-free D. lotus leaf extract was prepared and subjected to enzymatic hydrolysis with α-l-rhamnosidase under optimized conditions (pH 5.5, 55 °C, and 0.6 U/mL). Isoquercitrin production was monitored via high-performance liquid chromatography. Antioxidant and anti-inflammatory activities were assessed using the 2,2-diphenyl-1-picrylhydrazyl radical scavenging and lipoxygenase (LOX) inhibition assays, respectively. The enzymatic reaction resulted in complete conversion of 30 mM rutin into isoquercitrin within 180 min, increasing isoquercitrin content from 9.8 to 39.8 mM. The enzyme-converted extract exhibited significantly enhanced antioxidant activity, with a 48% improvement in IC₅₀ value compared with the untreated extract. Similarly, LOX inhibition increased from 39.2% to 48.3% after enzymatic conversion. Both extracts showed higher inhibition than isoquercitrin alone, indicating synergistic effects of other phytochemicals present in the extract. This study is the first to demonstrate that α-l-rhamnosidase-mediated conversion of rutin to isoquercitrin in D. lotus leaf extract significantly improves its antioxidant and anti-inflammatory activities. The enzymatically enhanced extract shows potential as a functional food or therapeutic ingredient. Full article

Background/Objectives: Most snakebite incidents in Latin America are caused by species of the Bothrops genus. Their venom induces severe local effects, against which antivenom therapy has limited efficacy. Metabolites derived from Coffea arabica have demonstrated anti-inflammatory and anticoagulant properties, suggesting their potential as therapeutic agents to inhibit the local effects induced by B. asper venom. Methods: Three enzymatic assays were performed: inhibition of the procoagulant and amidolytic activities of snake venom serine proteinases (SVSPs); inhibition of the proteolytic activity of snake venom metalloproteinases (SVMPs); and inhibition of the catalytic activity of snake venom phospholipases A₂ (PLA_2s). Additionally, molecular docking studies were conducted to propose potential inhibitory mechanisms of the metabolites chlorogenic acid, caffeine, and caffeic acid. Results: Green and roasted coffee extracts partially inhibited the enzymatic activity of SVSPs and SVMPs. Notably, the green coffee extract, at a 1:20 ratio, effectively inhibited PLA₂ activity. Among the individual metabolites tested, partial inhibition of SVSP and PLA₂ activities was observed, whereas no significant inhibition of SVMP proteolytic activity was detected. Chlorogenic acid was the most effective metabolite, significantly prolonging plasma coagulation time and achieving up to 82% inhibition at a concentration of 62.5 μM. Molecular docking analysis revealed interactions between chlorogenic acid and key active site residues of SVSP and PLA₂ enzymes from B. asper venom. Conclusions: The roasted coffee extract demonstrated the highest inhibitory effect on venom toxins, potentially due to the formation of bioactive compounds during the Maillard reaction. Molecular modeling suggests that the tested inhibitors may bind to and occupy the substrate-binding clefts of the target enzymes. These findings support further in vivo research to explore the use of plant-derived polyphenols as adjuvant therapies in the treatment of snakebite envenoming. Full article

Colorectal cancer (CRC) remains a predominant cause of global cancer-related mortality, highlighting the pressing demand for innovative therapeutic strategies. Natural polysaccharides have emerged as promising candidates in cancer research due to their multifaceted anticancer mechanisms and tumor-suppressive potential across diverse malignancies. In this study, we enzymatically extracted a polysaccharide, named ERPP, from Ruditapes philippinarum and comprehensively evaluated its anti-colorectal cancer activity. We conducted in vitro assays, including CCK-8 proliferation, clonogenic survival, scratch wound healing, and Annexin V-FITC/PI apoptosis staining, and the results demonstrated that ERPP significantly inhibited HT-29 cell proliferation, suppressed colony formation, impaired migratory capacity, and induced apoptosis. JC-1 fluorescence assays provided further evidence of mitochondrial membrane potential (MMP) depolarization, as manifested by a substantial reduction in the red/green fluorescence ratio (from 10.87 to 0.35). These antitumor effects were further validated in vivo using a zebrafish HT-29 xenograft model. Furthermore, ERPP treatment significantly attenuated tumor angiogenesis and downregulated the expression of the vascular endothelial growth factor A (Vegfaa) gene in the zebrafish xenograft model. Mechanistic investigations revealed that ERPP primarily activated the mitochondrial apoptosis pathway. RT-qPCR analysis showed an upregulation of the pro-apoptotic gene Bax and a downregulation of the anti-apoptotic gene Bcl-2, leading to cytochrome c (CYCS) release and caspase-3 (CASP-3) activation. Additionally, ERPP exhibited potent antioxidant capacity, achieving an 80.2% hydroxyl radical scavenging rate at 4 mg/mL. ERPP also decreased reactive oxygen species (ROS) levels within the tumor cells, thereby augmenting anticancer efficacy through its antioxidant activity. Collectively, these findings provide mechanistic insights into the properties of ERPP, underscoring its potential as a functional food component or adjuvant therapy for colorectal cancer management. Full article

Tuberaria lignosa (Sweet) Samp. (Cistaceae) is a herbaceous species native to southwestern Europe, traditionally used to treat wounds, ulcers, and inflammatory or infectious skin conditions. This study aimed to characterize the phytochemical profile of its aqueous leaf extract and evaluate its skin-related in vitro biological activities. The phenolic composition was determined using UHPLC-HRMS/MS, HPLC-DAD, and quantitative colorimetric assays. Antioxidant activity was assessed against synthetic free radicals, reactive oxygen and nitrogen species, transition metals, and pro-oxidant enzymes. Enzymatic inhibition of tyrosinase, hyaluronidase, collagenase, and elastase were evaluated using in vitro assays. Cytocompatibility was tested on human keratinocytes and NIH/3T3 fibroblasts using MTT and resazurin assays, respectively, while wound healing was evaluated on NIH/3T3 fibroblasts using the scratch assay. Antifungal activity was investigated against several Candida and dermatophyte species, while antibiofilm activity was tested against Epidermophyton floccosum. The extract was found to be rich in phenolic compounds, accounting for nearly 45% of its dry weight. These included flavonoids, phenolic acids, and proanthocyanidins, with ellagitannins (punicalagin) being the predominant group. The extract demonstrated potent antioxidant, anti-tyrosinase, anti-collagenase, anti-elastase, and antidermatophytic activities, including fungistatic, fungicidal, and antibiofilm effects. These findings highlight the potential of T. lignosa as a valuable and underexplored source of bioactive phenolic compounds with strong potential for the development of innovative approaches for skin care and therapy. Full article

Ultraviolet B (UVB) irradiation drives skin photodamage, prompting exploration of natural therapeutics. This study investigated the reparative effects and mechanisms of crude Gastrodia elata polysaccharides (GP) on UVB-induced acute skin damage. GP was extracted from fresh G. elata via water extraction and alcohol precipitation. It is a homogeneous polysaccharide with a weight-average molecular weight of 808.863 kDa, comprising Ara, Glc, Fru, and GalA. Histopathological analysis revealed that topical application of GP on the dorsal skin of mice effectively restored normal physiological structure, suppressing epidermal hyperplasia and collagen degradation. Biochemical assays showed that GP significantly reduced the activities of MPO and MDA following UVB exposure while restoring the enzymatic activities of SOD and GSH, thereby mitigating oxidative stress. Moreover, GP treatment markedly upregulated the anti-inflammatory cytokines TGF-β and IL-10 and downregulated the pro-inflammatory mediators IL-6, IL-1β, and TNF-α, suggesting robust anti-inflammatory effects. Transcriptomics revealed dual-phase mechanisms: Early repair (day 5) involved GP-mediated suppression of hyper inflammation and accelerated necrotic tissue clearance via pathway network modulation. Late phase (day 18) featured enhanced anti-inflammatory, antioxidant, and tissue regeneration processes through energy-sufficient, low-inflammatory pathway networks. Through a synergistic response involving antioxidation, anti-inflammation, promotion of collagen synthesis, and acceleration of skin barrier repair, GP achieves comprehensive repair of UVB-induced acute skin damage. Our findings not only establish GP as a potent natural alternative to synthetic photoprotective agents but also reveal novel pathway network interactions governing polysaccharide-mediated skin regeneration. Full article

Background: In order to address complex scenarios in anti-doping science, especially in cases where an unintentional exposure of athletes to prohibited substances and a corresponding contamination of doping control samples at the collection event are argued, an understanding of tissue-specific drug metabolism is essential. Hence, in this study, the metabolic capacity of the seminal vesicle using in vitro assays was investigated. Methods: The aim was to assess whether selected doping-relevant substances—stanozolol, LGD-4033, GW1516, trimetazidine, and anastrozole—are metabolised in seminal vesicle cellular fractions (SV-S9) and how that metabolism compares to biotransformations induced by human liver S9 fractions (HL-S9). Liquid chromatography coupled to high-resolution/accurate mass spectrometry (LC HRAM MS) enabled the sensitive detection and identification of metabolites, revealing a limited metabolic activity of SV-S9. Results: For LGD-4033, GW1516, and trimetazidine, minor metabolic transformations were observed, whereas no metabolites of stanozolol or anastrozole were detected. Gene expression analysis using digital polymerase chain reaction (dPCR) confirmed transcripts of CYP2D6, CYP2E1, and CYP2C9 in SV-S9, though no enzymatic activity was detected. Gene expression and enzymatic activity in CYP3A4 and CYP1A2—major hepatic enzymes—were absent in SV-S9. Conclusions: Overall, these pilot study results suggest that the seminal vesicle has only a low capacity for xenobiotic metabolism, which translates into a limited role in the biotransformation of drugs and, hence, the metabolic pattern. Full article

Clinacanthus nutans (Burm.f.) Lindau is a Southeast Asian medicinal plant traditionally used for treating skin inflammation and infections. This study evaluated its wound-healing potential through anti-inflammatory, cytoprotective, and antiviral mechanisms. HPLC-DAD analysis identified schaftoside as the major flavonoid in the 95% ethanolic leaf extract. In the lipopolysaccharide (LPS)-stimulated murine macrophage cell line (RAW 264.7), both C. nutans extract (5 and 50 μg/mL) and its flavonoid schaftoside (5 and 20 μg/mL) significantly downregulated the expression of pro-inflammatory genes, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and prostaglandin E₂ (PGE₂), under both pre-treatment and post-treatment conditions. ELISA confirmed dose-dependent inhibition of human COX-2 enzymatic activity, reaching up to 99.3% with the extract and 86.9% with schaftoside. In the endothelial cell models (CCL-209), the extract exhibited low cytotoxicity and effectively protected cells from LPS-induced apoptosis, preserving vascular integrity critical to tissue regeneration. Antiviral assays demonstrated suppression of HSV-2 replication, particularly during early infection, which may help prevent infection-related delays in wound healing. Collectively, these findings suggest that C. nutans and schaftoside promote wound repair by attenuating inflammatory responses, supporting endothelial survival, and controlling viral reactivation. These multifunctional properties highlight their potential as natural therapeutic agents for enhancing wound-healing outcomes. Full article

Inhibition of CK2 and/or PIM-1 kinases has been shown to induce apoptosis in a variety of cancer cell lines, underscoring their potential as valuable targets in anti-cancer drug development. In this study, a series of N-substituted derivatives of 4,5,6,7-tetrabromo-1H-benzimidazole, including 2-oxopropyl/2-oxobutyl substituents and their respective hydroxyl analogues, were synthesized and evaluated for anti-cancer activity. The compounds’ ability to inhibit CK2α and PIM-1 kinases was assessed through enzymatic assays, complemented by comprehensive in silico enzyme–substrate docking analyses. Cytotoxicity was evaluated using the MTT assay in human cancer cell lines—including acute lymphoblastic leukemia (CCRF-CEM) and breast cancer (MCF-7, MDA-MB-231)—as well as in normal Vero cells. Apoptosis induction in the two most responsive cell lines (CCRF-CEM and MCF-7) was further examined using flow cytometry-based assays, including annexin V binding, mitochondrial membrane potential disruption, caspase-3 activation, and cell cycle analysis. Intracellular inhibition of CK2 and PIM-1 kinases was confirmed in CCRF-CEM and MCF-7 cells using Western blot and phospho-flow cytometry. Among the synthesized compounds, we identified a novel TBBi derivative exhibiting pronounced pro-apoptotic activity and the ability to inhibit PIM-1 kinase intracellularly. These findings support the hypothesis that PIM-1 kinase represents a promising molecular target for the treatment of leukemia. Full article

In the present study, the primary by-products of the hemp-seed oil process—hemp seed cake flour and hemp seed protein concentrate—underwent enzymatic hydrolysis using proteases and carbohydrases, either individually or in combination. The effectiveness of these enzymatic treatments in releasing bioactive compounds was evaluated by assessing the antioxidant and anti-inflammatory properties of the aqueous extracts of both hydrolysed and untreated hemp by-products. The aim was to explore their potential senotherapeutic properties and promote their application as dietary supplements. Secondary metabolites such as flavonoids, phenolic acids, and catechins were analysed using high-performance liquid chromatography. Total phenolic, flavonoid, and protein contents were determined using spectrophotometric methods. Scavenging activity (2,2-Diphenyl-1-picrylhydrazyl scavenging assay (DPPH assay)), antioxidant power (Ferric reducing antioxidant power assay (FRAP assay)), and lipid peroxidation-reducing activity (thiobarbituric acid-reactive substance analysis) were assessed through in vitro assays. Possible anti-inflammatory effects were evaluated by assessing haemolysis inhibition. The impact of extracts on albumin glycation induced by exposure to fructose was also determined. To assess the toxicity of extracts, a zebrafish larvae model was employed. All extracts contained significant amounts of phenolic compounds, flavonoids, and proteins, and they exhibited notable activities in reducing lipid peroxidation and stabilising erythrocyte cell membranes. However, they did not significantly influence protein glycation (the glycation inhibition was only in the range of 15–40%). Our research demonstrates the substantial health-promoting potential, including senescence delay, of aqueous extracts from by-products of the hemp-seed oil process, which are available in large quantities and can serve as valuable supplements to support the health of animals, including humans, rather than being discarded as waste from oil production. Full article

Background/Objective: The expression of human steroid sulfatase (STS) is upregulated in castration-resistant prostate cancer (CRPC) and is associated with resistance to anti-androgen drugs, such as enzalutamide (Enza) and abiraterone (Abi). Despite the known link between STS overexpression and therapeutic unresponsiveness, the mechanism by which STS confers this phenotype remains incompletely understood. In this study, we sought to understand how STS induces treatment resistance in advanced prostate cancer (PCa) cells by exploring its role in altering mitochondrial activity. Methods: To examine the effects of increased STS expression on mitochondrial respiration and programming, we performed RNA sequencing (RNA-seq) analysis, the Seahorse XF Mito Stress Test, and a mitochondrial Complex I enzyme activity assay in STS-overexpressing cells (C4-2B STS) and in enzalutamide-resistant CPRC cells (C4-2B MDVR). We employed SI-2, the specific chemical inhibitor of STS, on C4-2B STS and C4-2B MDVR cells and evaluated STS activity inhibition on mitochondrial molecular pathways and mitochondrial respiration. Lastly, we examined the effects of dehydroepiandrosterone sulfate (DHEAS) supplementation on C4-2B STS organoids. Results: We present evidence from the transcriptomic profiling of C4-2B STS cells that there are enriched metabolic pathway signatures involved in oxidative phosphorylation, the electron transport chain, and mitochondrial organization. Moreover, upon STS inhibition, signaling in the electron transport chain and mitochondrial organization pathways is markedly attenuated. Findings from the Seahorse XF Mito Stress Test and mitochondrial Complex I enzyme activity assay demonstrate that STS overexpression increases mitochondrial respiration, whereas the inhibition of STS by SI-2 significantly reduces the oxygen consumption rate (OCR) and Complex I enzyme activity in C4-2B STS cells. Similarly, an increased OCR and electron transport chain Complex I enzymatic activity are observed in C4-2B MDVR cells and a decreased OCR upon SI-2 inhibition. Lastly, we show that STS overexpression promotes organoid growth upon DHEAS treatment. Conclusions: Our study demonstrates STS as a key driver of metabolic reprogramming and flexibility in advanced prostate cancer. Disrupting enhanced mitochondrial respiration via STS presents a promising strategy in improving CRPC treatment. Full article

Snakebite is a significant global public health challenge, and the limited application of antivenom has driven the exploration of novel therapies. Combination therapy using small-molecule drugs targeting phospholipases A₂ (PLA2) and metalloproteinases (SVMP) in venom shows great potential. Although Rhamnetin and RXP03 exhibit notable anti-phospholipase and anti-metalloproteinase activities, respectively, their antiophidic potential remains poorly explored. This study aims to evaluate the inhibitory effects of Rhamnetin and RXP03 on snake venom toxicity. Methodologically, we conducted in vitro enzymatic assays to quantify PLA₂/SVMP inhibition, murine models of envenomation (subcutaneous/intramuscular venom injection) to assess local tissue damage and systemic toxicity, and histopathological/biochemical analyses. In vitro experiments demonstrated that Rhamnetin effectively inhibited PLA₂ activity while RXP03 showed potent suppression of SVMP activity, with their combination significantly reducing venom-induced hemorrhagic activity. In murine models, the combined therapy markedly alleviated venom-triggered muscle toxicity and ameliorated oxidative stress. Furthermore, the combination enhanced motor performance and survival rate in mice, improved serum biochemical parameters, corrected coagulation disorders, and attenuated pathological damage in liver, kidney, heart, and lung tissues. This research demonstrates that dual-targeted therapy against metalloproteinases and phospholipases in snake venom can effectively prevent a series of injuries caused by snake venom. Collectively, the combined application of Rhamnetin and RXP03 exhibits significant inhibitory effects on a variety of venom-induced toxicities, providing pharmacological evidence for the development of antivenom therapies. However, the efficacy validation in this study was limited to murine models, and there is a discrepancy with clinical needs for delayed treatment in real-world envenomation scenarios. Despite these limitations, the findings provide robust preclinical evidence supporting the Rhamnetin–RXP03 combination therapy as a cost-effective, broad-spectrum antivenom strategy. Future studies are required to optimize dosing regimens and evaluate clinical translatability. Full article

Background and Objectives: Acute ulcerative colitis is characterized by excessive mucosal inflammation and epithelial disruption, often driven by dysregulated cytokine and immune signaling. Indoximod (1-methyl-DL-tryptophan), although not a direct enzymatic inhibitor, modulates the indoleamine 2,3-dioxygenase (IDO) pathway and has been reported to exert immunoregulatory effects in various models of inflammation. This study aimed to evaluate the protective effects of Indoximod in an acetic acid-induced colitis model in rats, focusing on histopathological changes and inflammatory mediators. Materials and Methods: Thirty male Wistar albino rats were randomly assigned to three groups (n = 10 per group): Group 1 (Control) received 0.9% saline oral gavage; Group 2 (Colitis) received intrarectal 4% acetic acid to induce colitis and were then treated with saline; Group 3 (Colitis + Indoximod) received 4% acetic acid followed by oral gavage administration of Indoximod (30 mg/kg) for 15 consecutive days. Histopathological evaluation of colonic tissues was performed using hematoxylin and eosin (H&E) staining. Colonic expression of Toll-like receptor 4 (TLR4) and plasma levels of tumor necrosis factor-alpha (TNF-α), pentraxin-3 (PTX-3), and platelet-activating factor (PAF) were quantified using enzyme-linked immunosorbent assay (ELISA). Results: Acetic acid-induced colitis significantly increased mucosal damage, TLR4 expression, and circulating levels of TNF-α, PTX-3, and PAF compared with controls (p < 0.001). Indoximod treatment markedly reduced histological injury and significantly suppressed TLR4 and TNF-α levels (p < 0.01), along with partial reductions in PTX-3 (p < 0.05). However, PAF levels remained elevated despite treatment, indicating limited efficacy in PAF-associated pathways. Conclusions: Indoximod exhibited anti-inflammatory effects in this acute colitis model, likely by downregulating key proinflammatory mediators. Full article

Background/Objectives: Studies have shown that Atractylenolide I (AT-I) can exert anti-inflammatory and anti-oxidative effects, protecting against the development of various kinds of cardiovascular diseases. However, whether AT-I prevents nicotine-induced atherogenesis is unknown. This study was designed to explore the effects of AT-I on nicotine-induced macrophage pyroptosis and the progression of atherosclerosis. Methods: RT-qPCR and Western blot were used to detect the mRNA and protein levels of TXNIP and pyroptosis-related factors in THP-1-derived macrophages. ELISA was used to detect the secretion of pro-inflammatory cytokines. Hoechst/PI double-staining assay was used to assess plasma membrane integrity. The ROS assay kit, LDH release assay kit, and caspase-1 activity assay kit were used to detect ROS production, LDH release, and caspase-1 activity. Oil Red O, HE, and Masson staining were used to evaluate lipid accumulation, lesion size, and plaque stability in HFD-fed apoE^−/− mice. Results: AT-I treatment significantly decreased pyroptosis-related factors expression, disrupted plasma membrane integrity, and down-regulated pro-inflammatory cytokines secretion, thereby inhibiting nicotine-induced pyroptosis of THP-1-derived macrophages. In addition, AT-I decreased ROS production and the expression of TLR4 and TXNIP. Lentivirus overexpression of TLR4 or TXNIP, or pre-treatment with ROS agonist, mainly reversed the anti-pyroptotic effects of AT-I in nicotine-treated THP-1-derived macrophages. Additionally, administering AT-I to HFD-fed apoE^−/− mice markedly decreased nicotine-induced up-regulation of pyroptosis-related proteins in the aortas. Enzymatic methods and ELISA assay suggested that AT-I improved dyslipidemia and inflammation in vivo. Oil Red O, HE, and Masson staining showed that AT-I alleviated lipid accumulation, decreased plaque size, and increased plaque stability. Conclusions: Taken together, AT-I can be regarded as a potential phytomedicine that protects against macrophage pyroptosis and atherosclerosis triggered by nicotine. Full article

Background: KRAS-G12D mutations drive 20–50% of pancreatic/biliary cancers yet remain challenging to target due to GTP-pocket conservation and high cellular GTP levels. While allosteric inhibitors targeting the SWII pocket (e.g., MRTX1133) show promise, limited chemical diversity and paradoxical cellular/enzymatic activity relationships necessitate the exploration of novel scaffolds. This study aims to develop KRAS-G12D inhibitors and PROTACs to offer a selection of new chemical entities through systematic structure–activity optimization and evaluate their therapeutic potential through PROTAC derivatization. Methods: Eleven compounds featuring heterocyclic cores (pyrimidine/pyrido[4,3-d]pyrimidine/5,6,7,8-tetrahydroprodo[3,4-d]pyrimidine) were designed via structure-based drug design. Antiproliferative activity against KRAS-G12D (Panc1), KRAS-G13D (HCT116) and wild-type (A549) cells was assessed using the CCK-8 assay. KRAS-G12D enzymatic inhibition was measured using a GTPase activity assay. Molecular docking simulations (Sybyl 2.0; PDB:7RPZ) elucidated binding modes. Two PROTACs were synthesized from lead compounds by conjugating E3 ligase linkers. All the novel inhibitors and PROTACs were characterized by means of NMR or HRMS. Results: Compound 10c demonstrated selective anti-proliferation in Panc1 cells (IC₅₀ = 1.40 μM) with 4.9-fold greater selectivity over wild-type cells, despite weak enzymatic inhibition (IC₅₀ > 10 μM). Docking revealed critical hydrogen bonds between its protonated 3,8-diazabicyclo[3.2.1]octane moiety and Asp12/Gly60. The enzymatic inhibitor 10k showed potent KRAS-G12D inhibition (IC₅₀ = 0.009 μM) through homopiperazine-mediated interactions with Glu92/His95. Derived PROTACs 26a/b exhibited reduced potency (IC₅₀ = 3–5 μM vs. parental 10k: 2.22 μM), potentially due to impaired membrane permeability. Conclusions: Eleven novel KRAS-G12D inhibitors with a seven-membered ring pharmacophore were synthesized. Compound 10c showed strong anti-proliferative activity, while 10k exhibited potent enzymatic inhibition. Two PROTACs were designed but showed no clear advantage over 10k. This study provides valuable insights for KRAS-targeted drug development. Full article