Computational exploration of Withania coagulans-derived natural inhibitors targeting HMG-CoA reductase: A pharmacokinetic, docking and molecular dynamics study for cholesterol regulation in Mus musculus.

Cholesterol is an essential component of cell membranes and is the immediate precursor of steroid hormones and bile acids. However, in excessive amounts, cholesterol becomes an important risk factor for cardiovascular disease, as demonstrated in clinical trials from the Framingham Heart Study [1–3] and the Multiple Risk Factor Intervention Trial [4, 5]. Although dietary cholesterol can contribute to changes in serum cholesterol levels, more than two-thirds of the body's cholesterol is synthesized in the liver. Therefore, inhibition of hepatic cholesterol biosynthesis has emerged as the therapeutic target of choice for reducing serum cholesterol levels. The rate-limiting enzyme in cholesterol biosynthesis in the liver is 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase [6], which catalyzes the conversion of HMG-CoA to mevalonic acid [7] (Fig. 1).

Background: Dyslipidemia is a major modifiable risk factor for cardiovascular diseases, the leading cause of mortality worldwide. The consolidation of interest in plant-based interventions has Spurred further examination of traditional medicinal herbs for their effect on lipid levels. Fenugreek (Trigonella foenum-graecum), an herb found in the traditional Ayurvedic and Chinese matrix, has emerged as a promising functional food. This is based on its phytochemical profile, in addition to its cardioprotective and health-promoting functions. Objective: This review sought to evaluate the lipid-lowering effects of fenugreek, describe potential biochemical and pharmacological mechanisms, and evaluate the clinical relevance of fenugreek is dyslipidemia and other metabolic states Methods: A comprehensive literature search was conducted using PubMed, Scopus, and Google Scholar, to identify relevant preclinical studies, clinical trials, and imaging and mechanistic studies published in the past 20 years. The key inclusion model was studies examining the effects of fenugreek seed, extract, or derived compounds on lipid profiles and cardiovascular characteristics in human and non-human subjects. Results: The chemical analysis revealed that fenugreek seeds are high in soluble dietary fiber (galactomannan), and abundant bioactive constituents, such as, steroidal saponins (e.g., diosgenin), flavonoids, polyphenols, alkaloids (e.g., trigonelline), and amino acids. Such constituents exert numerous lipid-modulating properties, such as: inhibit cholesterol absorption in the intestine; increase bile acid secretion; increase lipolytic enzymes; and downregulate the hepatic HMG-CoA reductase, which is the rate-limiting enzyme in cholesterol biosynthesis. Animal studies show that supplementation with fenugreek has significantly lowered total cholesterol, low density lipoprotein- cholesterol (LDL-C) and triglycerides, while simultaneously raising high density lipoprotein-cholesterol (HDL-C). The relatively few human clinical trials have also shown some encouraging data but sample sizes are too small and the studies too short in duration for any statistically valid claims. However, some literature exists documenting favourable changes in lipid profiles associated with fenugreek in patients diagnosed with hyperlipidemia, type 2 diabetes and metabolic syndrome. Fenugreek was also found to be highly palatable and offer multiple forms of consumption i.e. whole seeds, defatted powder (flour), aqueous extracts and nutraceutical formulations; thus, supporting potential for promotion, uptake and long-term consumption as dietary therapy. Conclusion: Fenugreek shows significant potential as an affordable, natural tool for the prevention and treatment of dyslipidemia and cardiovascular disease risk. Its diverse lipid-lowering mechanisms, benign safety profile, and straightforward incorporation into the diet classify it as a candidate for both pharmacological and dietary components of cardiovascular care. However, the present evidence base is limited by methodologic challenges, differences in primary extract type, and a lack of large and well-designed randomized controlled trials. Future research should focus on developing standard doses of fenugreek combined with safety and effectiveness studies designed and executed in large clinical trials to enable evidence-based recommendations for its inclusion in evidence-based treatment protocols.

Differential sensitivity to toxicity of statins: A comparison of liver and muscle cell lines

Background Cardiovascular diseases comprehend a array of situations that disturb the heart and blood vessels, making them a principal cause of morbidity and mortality worldwide. Dyslipidemia, an imbalance in lipids, such as cholesterol, Low-Density Lipoprotein Cholesterol (LDL-C), triglycerides and High-Density Lipoprotein (HDL), is a significant risk factor for cardiovascular disease and contributes to numerous deaths annually. The development of cholesterol-degrading drugs, known as statins, began in the mid-1970s and their effects on intracellular processes have been well documented. Materials and Methods Fluvastatin, a statin widely used in cardiovascular disease, inhibits hydroxymethyl glutaryl-coenzyme A, a vital enzyme in cholesterol biosynthesis. This study aimed to discover new therapeutic drugs to prevent cardiovascular diseases by deriving new compounds from fluvastatin. Results The investigation involved several processes including derivative generation, molecular docking, ADME analysis, PASS prediction, rodent toxicity assessment and identification of possible adverse effects. After evaluating a vast dataset of 194 derivatives, compounds that met stringent criteria across all parameters were selected. Conclusion Two promising compounds from this filtered set were further assessed through Molecular Dynamics (MD) simulation and Principal Component Analysis (PCA), along with the control.

Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans.

Background: Cardiovascular diseases (CVDs) remain the leading cause of mortality worldwide, with dysregulation of cholesterol metabolism as a key contributor. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the rate-limiting enzyme in cholesterol biosynthesis, targeted effectively by statins. However, statins can cause adverse effects, highlighting the need for safer, natural alternatives. Gallic acid and chlorogenic acid, abundant phenolic compounds in Azadirachta indica leaves, have shown potential HMGR inhibitory activity. The objective was to evaluate the HMGR inhibitory potential of gallic acid and chlorogenic acid through molecular docking analysis, thereby elucidating their possible role as natural lipid-lowering agents. Methods: Molecular docking was performed using AutoDock Vina across seven potential binding sites of HMGR, identified from crystal structure analysis. Ligand structures were prepared from PubChem, and receptor refinement was conducted in BIOVIA Discovery Studio. Binding affinities and ligand–protein interactions were analyzed to assess inhibitory potential. Results: Binding energies for gallic acid ranged from –3.7 to –6.0 kcal/mol, with strongest binding at Site 5 (–6.0 kcal/mol), primarily stabilized by hydrophobic (Pi–Sigma, Pi–Alkyl) and electrostatic interactions (Pi–Anion, Pi–Cation). Chlorogenic acid exhibited higher binding affinities across sites (–4.3 to –7.3 kcal/mol), with Sites 5 (–7.2 kcal/mol) and 7 (–7.3 kcal/mol) showing the strongest interactions, dominated by hydrophobic alkyl–alkyl contacts. Overall, chlorogenic acid demonstrated stronger site-specific binding than gallic acid, though both showed weaker binding compared to statins. Discussion & Conclusion: Chlorogenic acid exhibits greater affinity for HMGR than gallic acid, suggesting its potential as a moderate natural HMGR inhibitor. These findings warrant further in vitro and in vivo studies to validate their cholesterol-lowering potential and explore their use as safer alternatives or adjuncts to statin therapy.

Breast cancer (BC) remains a leading cause of cancer-related mortality in women. The oncoprotein MDM2 negatively regulates the tumor suppressor p53, and its overexpression in BC promotes tumor progression and resistance to therapy. Targeting the MDM2-p53 interaction represents a promising therapeutic approach. However, many existing MDM2 inhibitors suffer from poor pharmacokinetics and off-target toxicity, necessitating the discovery of novel, more selective alternatives. This study aims to identify natural terpenoid compounds with potent MDM2 inhibitory potential through computational approaches. A library of 398 natural terpenoids was sourced from the NPACT database and filtered based on Lipinski's Rule of Five. A two-stage docking strategy was applied: 1) rigid protein-flexible ligand docking to screen for high-affinity binders, followed by 2) ensemble docking using multiple MDM2 conformations derived from molecular dynamics (MD) simulations. The top candidates were further evaluated for their pharmacokinetic and toxicity profiles using ADMET analysis. Finally, 150ns MD simulations and binding free energy (MM-PBSA) calculations were performed to assess the stability and strength of protein-ligand interactions. Three terpenoid compounds, olean-12-en-3-beta-ol, cabralealactone, and 27-deoxyactein demonstrated strong binding affinities toward MDM2 in ensemble docking studies. ADMET analysis confirmed their favorable pharmacokinetic properties. Further MD simulations indicated that these compounds formed highly stable complexes with MDM2. Notably, 27-deoxyactein exhibited the lowest binding free energy (-154.514kJ/mol), outperforming the reference inhibitor Nutlin-3a (-133.531kJ/mol), suggesting superior binding stability and interaction strength. Our findings highlight 27-deoxyactein as a promising MDM2 inhibitor with strong binding affinity, stability, and a favorable pharmacokinetic profile. This study provides a computational foundation for further experimental validation, supporting the potential of terpenoid-based MDM2 inhibitors in BC therapy.

BackgroundStatins are widely used cholesterol-lowering drugs that act by inhibiting HMGCoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent evidence suggests that statin use may be associated with a decreased risk for Alzheimer disease, although the mechanisms underlying this apparent risk reduction are poorly understood. One popular hypothesis for statin action is related to the drugs' ability to activate α-secretase-type shedding of the α-secretase-cleaved soluble Alzheimer amyloid precursor protein ectodomain (sAPPα). Statins also inhibit the isoprenoid pathway, thereby modulating the activities of the Rho family of small GTPases—Rho A, B, and C—as well as the activities of Rac and cdc42. Rho proteins, in turn, exert many of their effects via Rho-associated protein kinases (ROCKs). Several cell-surface molecules are substrates for activated α-secretase-type ectodomain shedding, and regulation of shedding typically occurs via activation of protein kinase C or extracellular-signal-regulated protein kinases, or via inactivation of protein phosphatase 1 or 2A. However, the possibility that these enzymes play a role in statin-stimulated shedding has been excluded, leading us to investigate whether the Rho/ROCK1 protein phosphorylation pathway might be involved.Methods and FindingsWe found that both atorvastatin and simvastatin stimulated sAPPα shedding from a neuroblastoma cell line via a subcellular mechanism apparently located upstream of endocytosis. A farnesyl transferase inhibitor also increased sAPPα shedding, as did a dominant negative form of ROCK1. Most conclusively, a constitutively active ROCK1 molecule inhibited statin-stimulated sAPPα shedding.ConclusionTogether, these data suggest that statins exert their effects on shedding of sAPPα from cultured cells, at least in part, by modulation of the isoprenoid pathway and ROCK1.

Introduction Giant cell arteritis (GCA) is a chronic granulomatous vasculitis preferentially targeting largeand medium-sized vessels in aged people. The inflammatory lesions eventually lead to vessel occlusion and 15% of patients develop cranial ischemic complications, particularly visual loss (1,2). The granulomatous nature of GCA lesions, with the frequent presence of multinucleated giant cells, has classically suggested a delayed-type hypersensitivity reaction but the potential triggering agents remain unknown (1–3). CD4 T cells infiltrating the vessel wall display a T helper type 1 functional differentiation with copious production of interferon (IFN ), a major cytokine in macrophage activation (3). Activated macrophages produce angiogenic factors and proinflammatory cytokines, such as interleukin-1 , tumor necrosis factor , and interleukin-6 (IL-6) (2,3). These mediators amplify the inflammatory response by inducing endothelial cell adhesion molecules for leukocytes and chemokines, and by generating new vessels through which additional leukocytes may subsequently invade the vessel wall (2,4–7). Macrophages also participate in tissue destruction by producing oxidative damage and secreting metalloproteases, and in tissue repair by secreting fibrogenic cytokines that eventually may lead to vessel occlusion with its ensuing ischemic complications (3). Corticosteroids are, at present, the treatment of choice for patients with GCA (1–3). Although their ability to modify the course of the disease or to cure it is questionable, corticosteroids induce dramatic functional changes in GCA lesions both in humans and in human arteritis– severe combined immunodeficient mouse chimeras (4,8,9). These functional changes result in a rapid relief of symptoms and prevention of ischemic complications. However, therapeutic requirements are highly variable among patients. Some patients achieve persistent remission of the disease within a few months, whereas others present recurrent relapses and need maintenance doses of corticosteroids for long periods of time. Sustained corticosteroid therapy has been associated with the development of dyslipidemia by inducing insulin resistance, increasing the hepatic synthesis of very lowdensity lipoproteins and triglyceride, enhancing the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, and inhibiting lipoprotein lipase. Moreover, studies in vitro have demonstrated that corticosteroids inhibit the activity of the low-density lipoprotein (LDL) receptor leading to an increase in LDL levels in patients (10–13). For that reason, some patients diagnosed with GCA receiving corticosteroid therapy may require lipidlowering agents during their followup. HMG-CoA reductase inhibitors, statins, are widely and effectively used as hypolipidemic agents. They competitively inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Treatment with statins results in a reduction of the cholesterol levels through a decrease in cholesterol synthesis and by increasing the expression of hepatic LDL receptors, which clear LDL and LDL precursors from the bloodstream (14). Numerous clinical trials have demonstrated that statins reduce coronary heart disease mortality and the incidence of cardiovascular events (14–16). Recently, it has been demonstrated that statins also have antiinflammatory properties, through various mechanisms (14). The effect of statins on the inflammatory component of atherosclerosis is considered to be an important mechanism through which statins reduce cardiovascular events and death (14,17,18). Furthermore, statins have demonstrated to be of therapeutic benefit in animal models of chronic inflammatory conditions (19– 21) and have been shown to reduce graft rejection in heart Supported by Ministerio de Ciencia y Tecnologia and Fondo Europeo de Desarrollo Regional (FEDER; SAF 0203307) and Generalitat de Catalunya (2001/SGR/00379). Dr. Garcia-Martinez was supported by a research award from Hospital Clinic. Dr. Cid was supported by a research award from IDIBAPS. Ana Garcia-Martinez, MD, Jose Hernandez-Rodriguez, MD, Josep M. Grau, MD, Maria C. Cid, MD: Hospital Clinic, University of Barcelona, Barcelona, Spain. Address correspondence to Maria C. Cid, MD, Department of Internal Medicine, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain. E-mail: mccid@clinic.ub.es. Submitted for publication June 18, 2003; accepted in revised form December 16, 2003. Arthritis & Rheumatism (Arthritis Care & Research) Vol. 51, No. 4, August 15, 2004, pp 674–678 DOI 10.1002/art.20541 © 2004, American College of Rheumatology

Endotoxin-lipoprotein hypothesis

Atopic dermatitis (AD) is a chronic, multifactorial inflammatory skin disease characterized by persistent pruritus, immune system dysregulation, and an increased expression of cyclooxygenase-2 (COX-2), an enzyme that plays a central role in the production of prostaglandins and the promotion of inflammatory responses. In this study, we employed a comprehensive computational pipeline to identify phytocompounds capable of inhibiting COX-2 activity, offering an alternative to traditional non-steroidal anti-inflammatory drugs. The African and Traditional Chinese Medicine natural product databases were subjected to molecular screening, which identified six top compounds, namely, Tophit1 (−16.528 kcal/mol), Tophit2 (−10.879 kcal/mol), Tophit3 (−9.760 kcal/mol), Tophit4 (−9.752 kcal/mol), Tophit5 (−8.742 kcal/mol), and Tophit6 (−8.098 kcal/mol), with stronger binding affinities to COX-2 than the control drug rofecoxib (−7.305 kcal/mol). Molecular dynamics simulations over 200 ns, combined with MM/GBSA binding free energy calculations, consistently identified Tophit1 and Tophit2 as the most stable complexes, exhibiting exceptional structural integrity and a strong binding affinity to the target protein. ADMET profiling via SwissADME and pkCSM validated the drug-likeness, oral bioavailability, and safety of the lead compounds, with no Lipinski rule violations and favorable pharmacokinetic and toxicity profiles. These findings underscore the therapeutic potential of the selected phytocompounds as novel COX-2 inhibitors for the management of atopic-prone skin and warrant further experimental validation.

Effect of dietary miso on the activities of various hepatic enzymes in lipid biosynthesis and degradation was examined in rats fed a cholesterol-free diet for 18 days. The inclusion of 20% desalted miso to the experimental diet significantly decreased the concentration of serum cholesterol and doubled the fecal excretion of acidic steroids without influencing that of neutral steroids. The dietary miso greatly increased the activities of the rate-limiting enzymes in cholesterol biosynthesis and degradation in liver microsomes. In contrast, dietary miso was ineffective in modulating activities of enzymes in fatty acid oxidation and glycerolipid synthesis. Microsomal lipid contents and compositions were uninfluenced by dietary miso. It was suggested that the miso contained the component (s) which specifically increase the facal excretion of bile acids. The enhanced bile acid excretion may be compensated by the increase in the catabolism of cholesterol to bile acids and reduce the serum cholesterol concentration as observed in the present study.

Despite important advances in the management of hypercholesterolemia in recent decades, many patients with lipid disorders remain unidentified or undertreated and so continue to have unfavorable levels of low-density lipoprotein (LDL) cholesterol and an increased risk for coronary events. The statins--which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis--have proved to be the most powerful pharmacologic agents for lowering serum lipids, and newer statins offer even greater efficacy than the agents introduced 10 to 15 years ago. Studies have shown that rosuvastatin, in late-stage development, is a very potent agent for the treatment of primary hypercholesterolemia, and that relatively low doses decrease LDL cholesterol levels to a greater extent than do similar doses of pravastatin, simvastatin, or atorvastatin as evaluated in separate clinical trials. Pitavastatin, in phase II trials, also has promise as a more potent drug than currently available statins. Because neither of these drugs has been approved for use in the United States, clinical trial results should be considered preliminary. In the future, agents that combine the actions of statins and nicotinic acid may achieve still greater LDL cholesterol reductions. Drugs that lower lipids via mechanisms other than inhibition of HMG-CoA reductase also offer promise. The newest addition to the roster of lipid-regulating agents is ezetimibe, a cholesterol absorption inhibitor that has been approved for use either alone or in combination with a statin. Agents in development include bile acid transport inhibitors and inhibitors of acyl CoA:cholesterol acyltransferase. More research will be needed to determine the full clinical potential of such approaches to the management of hypercholesterolemia.

Hypercholesterolemia is recognized as a major risk factor for cardiovascular diseases and was commonly treated with long-term synthetic drugs, which were costly and associated with side effects. Medicinal plants, such as menteng (Baccaurea racemosa) leaves, have been explored as potential natural alternatives. This study investigated the anti-cholesterol activity of different menteng leaf extract fractions in vitro, identified the bioactive compounds in the most active fraction using LC-MS/MS, and assessed their potential as drug candidates through an in-silico approach. The research involved stepwise fractionation with n-hexane, ethyl acetate, and n-butanol solvents. Cholesterol level was evaluated in-vitro using the Liebermann-Burchard method by reacting with acetic anhydride and sulfuric acid, and bioactive compounds were identified through LC-MS/MS analysis. Molecular docking, biological activity prediction, and toxicity analysis were also performed. The results showed that the n-butanol fraction exhibited the highest in vitro anti-cholesterol activity (EC50 = 45.16 mg/L), surpassing the ethanol extract and other fractions. LC-MS/MS analysis revealed 7 bioactive compounds, including flavonoids (quercitrin, apiin and vitexin), phenolics (nicotiflorin), alkaloids (indole), phenylpropanoids (umbelliferone), and coumarins. The in-silico study demonstrated that these compounds had strong binding affinities compared to control drugs. These findings suggested that menteng leaf extracts, particularly the n-butanol fraction, could serve as a natural anti-cholesterol agent. Further in vivo studies and clinical trials are needed to confirm their therapeutic efficacy. HIGHLIGHTS Menteng (Baccaurea racemosa) leaves exhibit strong anti-cholesterol activity, with the n-butanol fraction showing the highest effectiveness, achieving an EC50 value of 45.16 mg/L. This suggests its potential as a natural alternative for managing hypercholesterolemia. LC-MS/MS analysis identified seven bioactive compounds, including flavonoids (nicotiflorin, quercitrin, apiin, vitexin), alkaloids (indole), and phenylpropanoids (umbelliferone, coumarin). These compounds demonstrated strong binding affinity in molecular docking studies, comparable to standard cholesterol-lowering drugs. In silico studies revealed that menteng leaf bioactive compounds could effectively target HMG-CoA reductase, a key enzyme in cholesterol biosynthesis. The findings highlight the potential of these compounds as natural anti-cholesterol drug candidates, warranting further in vivo and clinical investigation. GRAPHICAL ABSTRACT

BackgroundHypercholesterolemia has posed a serious threat of heart diseases and stroke worldwide. Xanthine oxidase (XO), the rate-limiting enzyme in uric acid biosynthesis, is regarded as the root of reactive oxygen species (ROS) that generate atherosclerosis and cholesterol crystals. β-Hydroxy β-methylglutaryl-coenzyme A reductase (HMGR) is a rate-limiting enzyme in cholesterol biosynthesis. Although some commercially available enzyme inhibiting drugs have effectively reduced cholesterol levels, most of them have failed to meet potential drug candidates’ requirements. Here, we have carried out an in-silico analysis of secondary metabolites that have already shown good inhibitory activity against XO and HMGR in a wet lab setup.MethodsOut of 118 secondary metabolites reviewed, sixteen molecules inhibiting XO and HMGR were selected based on the IC50 values reported in in vitro assays. Further, receptor-based virtual screening was carried out against secondary metabolites using GOLD Protein-Ligand Docking Software, combined with subsequent post-docking, to study the binding affinities of ligands to the enzymes. In-silico ADMET analysis was carried out to explore their pharmacokinetic properties, followed by toxicity prediction through ProTox-II.ResultsThe molecular docking of amentoflavone (GOLD score 70.54, ∆G calc. = − 10.4 Kcal/mol) and ganomycin I (GOLD score 59.61, ∆G calc. = − 6.8 Kcal/mol) displayed that the drug has effectively bound at the competitive site of XO and HMGR, respectively. Besides, 6-paradol and selgin could be potential drug candidates inhibiting XO. Likewise, n-octadecanyl-O-α-D-glucopyranosyl (6′ → 1″)-O-α-D-glucopyranoside could be potential drug candidates to maintain serum cholesterol. In-silico ADMET analysis has shown that these sixteen metabolites were optimal within the categorical range compared to commercially available XO and HMGR inhibitors, respectively. Toxicity analysis through ProTox-II revealed that 6-gingerol, ganoleucoin K, and ganoleucoin Z are toxic for human use.ConclusionThis computational analysis supports earlier experimental evidence towards the inhibition of XO and HMGR by natural products. Further study is necessary to explore the clinical efficacy of these secondary molecules, which might be alternatives for the treatment of hypercholesterolemia.